Workplace Exposure Measurements of Emission from Industrial 3D Printing.

Particle and gaseous contaminants from industrial scale additive manufacturing (AM) machines were studied in three different work environments. Workplaces utilized powder bed fusion, material extrusion, and binder jetting techniques with metal and polymer powders, polymer filaments, and gypsum powder, respectively. The AM processes were studied from operator's point of view to identify exposure events and possible safety risks. Total number of particle concentrations were measured in the range of 10 nm to 300 nm from operator's breathing zone using portable devices and in the range of 2.5 nm to 10 µm from close vicinity of the AM machines using stationary measurement devices. Gas-phase compounds were measured with photoionization, electrochemical sensors, and an active air sampling method which were eventually followed by laboratory analyses. The duration of the measurements varied from 3 to 5 days during which the manufacturing processes were practically continuous. We identified several work phases in which an operator can potentially be exposed by inhalation (pulmonary exposure) to airborne emissions. A skin exposure was also identified as a potential risk factor based on the observations made on work tasks related to the AM process. The results confirmed that nanosized particles were present in the breathing air of the workspace when the ventilation of the AM machine was inadequate. Metal powders were not measured from the workstation air thanks to the closed system and suitable risk control procedures. Still, handling of metal powders and AM materials that can act as skin irritants such as epoxy resins were found to pose a potential risk for workers. This emphasizes the importance of appropriate control measures for ventilation and material handling that should be addressed in AM operations and environment.

Hopping Light Vat Photopolymerization for Multiscale Fabrication.

3D objects with features spanning from microscale to macroscale have various applications. However, the fabrication of such objects presents challenges to additive manufacturing (AM) due to the tradeoffs among manufacturable feature resolution, maximum build area, and printing speed. This paper presents a projection-based AM process called hopping light vat photopolymerization (HL-VPP) to address this critical barrier. The key idea of HL-VPP is to synchronize linear scanning projection with a galvo mirror's rotation. The projector moves continuously at a constant speed while periodically rotating a one-axis galvo mirror to compensate for the projector's linear movement so synchronized hopping motion can be achieved. By this means, HL-VPP can simultaneously achieve large-area (over 200 mm), fast-speed (scanning speed of 13.5 mm s-1 ), and high-resolution (10 µm pixel size) fabrication. The distinguishing characteristic of HL-VPP is that it allows for hundreds of times lower refresh rates without motion blur. Thus, HL-VPP decouples the fabrication efficiency limit imposed by the refresh rate and will enable super-fast curing in the future. This work will significantly advance VPP's use in applications that require macroscale part size with microscale features. The process has been verified by fabricating multiple multiscale objects, including microgrids and biomimetic structures.

Patient-Specific Bioimplants and Reconstruction Plates for Mandibular Defects: Production Workflow and In Vivo Large Animal Model Study.

A major challenge with extensive craniomaxillofacial bone reconstruction is the limited donor-site availability to reconstruct defects predictably and accurately according to the anatomical shape of the patient. Here, patient-specific composite bioimplants, consisting of cross-linked poly(trimethylene carbonate) (PTMC) networks and ß-tricalcium phosphate (ß-TCP), are tested in vivo in twelve Göttingen minipigs in a large mandibular continuity defect model. The 25 mm defects are supported by patient-specific titanium reconstruction plates and receive either osteoconductive composite bioimplants (PTMC+TCP), neat polymer network bioimplants (PTMC), autologous bone segments (positive control), or are left empty (negative control). Postoperatively, defects treated with bioimplants show evident ossification at 24 weeks. Histopathologic evaluation reveals that neat PTMC bioimplant surfaces are largely covered with fibrous tissue, while in the PTMC+TCP bioimplants, bone attached directly to the implant surface shows good osteoconduction and histological signs of osteoinductivity. However, PTMC+TCP bioimplants are associated with high incidence of necrosis and infection, possibly due to rapid resorption and/or particle size of the used ß-TCP. The study highlights the importance of testing bone regeneration implants in a clinically relevant large animal model and at the in situ reconstruction site, since results on small animal models and studies in nonloadbearing areas do not translate directly.

Endodormancy release in Norway spruce grafts representing trees of different ages.

Studies addressing endodormancy release in adult trees are usually carried out using twigs detached from the trees in the experiments. Potential problems caused by cutting the root-shoot connection when detaching the twigs can be avoided by using grafts as the experimental material. We studied the effects of chilling on the endodormancy release in Norway spruce (Picea abies (L.) Karst.) grafts where twigs of 16-, 32- and 80-year-old trees were used as the scions. The grafts were first exposed to chilling in natural conditions and then samples of them were transferred at intervals to a regrowth test in forcing conditions in a greenhouse. The bud burst percentage, BB%, in the forcing conditions generally increased from zero to near 100% with increasing previous chilling accumulation from mid-October until mid-November, indicating that endodormancy was released in almost all of the grafts by mid-November. The days to bud burst, DBB, decreased in the forcing conditions with successively later transfers until the next spring. Neither BB% nor DBB was dependent on the age of the scion. However, in the early phase of ecodormancy release, the microscopic internal development of the buds was more advanced in the grafts representing the 16-year-old than in those representing the 32- or 80-year-old trees. In conclusion, our findings suggest that no major change in the environmental regulation of endodormancy release in Norway spruce takes place when the trees get older. Taken together with earlier findings with Norway spruce seedlings, our results suggest that regardless of the seedling or tree age, the chilling requirement of endodormancy release is met in late autumn. The implications of our findings for Norway spruce phenology under climatic warming and the limitations of our novel method of using grafts as a proxy of trees of different ages are discussed.

Techno-Economic Prospects and Desirability of 3D Food Printing: Perspectives of Industrial Experts, Researchers and Consumers.

3D food printing is an emerging food technology innovation that enables the personalization and on-demand production of edible products. While its academic and industrial relevance has increased over the past decade, the functional value of the technology remains largely unrealized on a commercial scale. This study aimed at updating the business outlook of 3D food printing so as to help entrepreneurs and researchers in the field to channel their research and development (R&D) activities. A three-phase mixed methods approach was utilized to gain perspectives of industrial experts, researchers, and potential consumers. Data were collected from two sets of interviews with experts, a survey with experts, and consumer focus group discussions. The results gave insights into key attributes and use cases for a 3D food printer system, including the techno-economic feasibility and consumer desirability of identified use cases. A business modelling workshop was then organized to translate these results into three refined value propositions for 3D food printing. Both the experts and consumers found personalized nutrition and convenience to be the most desirable aspects of 3D food printing. Accordingly, business models related to 3D printed snacks/meals in semi-public spaces such as fitness centers and hospitals were found to offer the highest business potential. While the technology might be mature enough at component level, the successful realization of such high-reward models however would require risk-taking during the developmental phase.

Improved Bone Regeneration in Rabbit Bone Defects Using 3D Printed Composite Scaffolds Functionalized with Osteoinductive Factors.

Large critical size bone defects are complicated to treat, and in many cases, autografts become a challenge due to size and availability. In such situations, a synthetic bone implant that can be patient-specifically designed and fabricated with control over parameters such as porosity, rigidity, and osteogenic cues can act as a potential synthetic bone substitute. In this study, we produced photocuring composite resins with poly(trimethylene carbonate) containing high ratios of bioactive ceramics and printed porous 3D composite scaffolds to be used as bone grafts. To enhance the overall surface area available for cell infiltration, the scaffolds were also filled with a macroporous cryogel. Furthermore, the scaffolds were functionalized with osteoactive factors: bone morphogenetic protein and zoledronic acid. The scaffolds were evaluated in vitro for biocompatibility and for functionality in vivo in critical bone defects (∼8 mm) in two clinically relevant rabbit models. These studies included a smaller study in rabbit tibia and a larger study in the rabbit cranium. It was observed that the bioactive molecule-functionalized 3D printed porous composite scaffolds provide an excellent conductive surface inducing higher bone formation and improved defect healing in both critical size long bones and cranial defects. Our findings provide strong evidence in favor of these composites as next generation synthetic bone substitutes.

Biomimetic Photocurable Three-Dimensional Printed Nerve Guidance Channels with Aligned Cryomatrix Lumen for Peripheral Nerve Regeneration.

Repair and regeneration of critically injured peripheral nerves is one of the most challenging reconstructive surgeries. Currently available and FDA approved nerve guidance channels (NGCs) are suitable for small gap injuries, and their biological performance is inferior to that of autografts. Development of biomimetic NGCs with clinically relevant geometrical and biological characteristics such as topographical, biochemical, and haptotactic cues could offer better regeneration of the long-gap complex nerve injuries. Here, in this study, we present the development and preclinical analysis of three-dimensional (3D) printed aligned cryomatrix-filled NGCs along with nerve growth factor (NGF) (aCG + NGF) for peripheral nerve regeneration. We demonstrated the application of these aCG + NGF NGCs in the enhanced and successful regeneration of a critically injured rat sciatic nerve in comparison to random cryogel-filled NGCs, multichannel and clinically preferred hollow conduits, and the gold standard autografts. Our results indicated similar effect of the aCG + NGF NGCs viz-a-viz that of the autografts, and they not only enhanced the overall regenerated nerve physiology but could also mimic the cellular aspects of regeneration. This study emphasizes the paradigm that these biomimetic 3D printed NGCs will lead to a better functional regenerative outcome under clinical settings.

Implementation of Industrial Additive Manufacturing: Intelligent Implants and Drug Delivery Systems.

The purpose of this study is to demonstrate the ability of additive manufacturing, also known as 3D printing, to produce effective drug delivery devices and implants that are both identifiable, as well as traceable. Drug delivery devices can potentially be used for drug release in the direct vicinity of target tissues or the selected medication route in a patient-specific manner as required. The identification and traceability of additively manufactured implants can be administered through radiofrequency identification systems. The focus of this study is to explore how embedded medication and sensors can be added in different additive manufacturing processes. The concept is extended to biomaterials with the help of the literature. As a result of this study, a patient-specific drug delivery device can be custom-designed and additively manufactured in the form of an implant that can identify, trace, and dispense a drug to the vicinity of a selected target tissue as a patient-specific function of time for bodily treatment and restoration.

Nanofibrillar cellulose-alginate hydrogel coated surgical sutures as cell-carrier systems.

Hydrogel nanomaterials, especially those that are of non-human and non-animal origins, have great potential in biomedical and pharmaceutical sciences due to their versatility and inherent soft-tissue like properties. With the ability to simulate native tissue function, hydrogels are potentially well suited for cellular therapy applications. In this study, we have fabricated nanofibrillar cellulose-alginate (NFCA) suture coatings as biomedical devices to help overcome some of the limitations related to cellular therapy, such as low cell survivability and distribution out of target tissue. The addition of sodium alginate 8% (w/v) increased the NFCA hydrogel viscosity, storage and loss moduli by slightly under one order of magnitude, thus contributing significantly to coating strength. Confocal microscopy showed nearly 100% cell viability throughout the 2-week incubation period within and on the surface of the coating. Additionally, typical morphologies in the dual cell culture of spheroid forming HepG2 and monolayer type SK-HEP-1 were observed. Twelve out of 14 NFCA coated surgical sutures remained intact during the suturing operation with various mice and rat tissue; however, partial peeling off was observed in 2 of the coated sutures. We conclude that NFCA suture coatings could perform as cell-carrier systems for cellular based therapy and post-surgical treatment.

Human stem cell decorated nanocellulose threads for biomedical applications.

Upon surgery, local inflammatory reactions and postoperative infections cause complications, morbidity, and mortality. Delivery of human adipose mesenchymal stem cells (hASC) into the wounds is an efficient and safe means to reduce inflammation and promote wound healing. However, administration of stem cells by injection often results in low cell retention, and the cells deposit in other organs, reducing the efficiency of the therapy. Thus, it is essential to improve cell delivery to the target area using carriers to which the cells have a high affinity. Moreover, the application of hASC in surgery has typically relied on animal-origin components, which may induce immune reactions or even transmit infections due to pathogens. To solve these issues, we first show that native cellulose nanofibers (nanofibrillated cellulose, NFC) extracted from plants allow preparation of glutaraldehyde cross-linked threads (NFC-X) with high mechanical strength even under the wet cell culture or surgery conditions, characteristically challenging for cellulosic materials. Secondly, using a xenogeneic free protocol for isolation and maintenance of hASC, we demonstrate that cells adhere, migrate and proliferate on the NFC-X, even without surface modifiers. Cross-linked threads were not found to induce toxicity on the cells and, importantly, hASC attached on NFC-X maintained their undifferentiated state and preserved their bioactivity. After intradermal suturing with the hASC decorated NFC-X threads in an ex vivo experiment, cells remained attached to the multifilament sutures without displaying morphological changes or reducing their metabolic activity. Finally, as NFC-X optionally allows facile surface tailoring if needed, we anticipate that stem-cell-decorated NFC-X opens a versatile generic platform as a surgical bionanomaterial for fighting postoperative inflammation and chronic wound healing problems.

Internal development of vegetative buds of Norway spruce trees in relation to accumulated chilling and forcing temperatures.

The timing of budburst of temperate trees is known to be controlled by complicated interactions of temperature and photoperiod. To improve the phenological models of budburst, better knowledge of the internal bud development preceding budburst in relation to environmental cues is needed. We studied the effect of accumulated chilling and forcing temperatures on the internal development of vegetative buds preceding budburst in Norway spruce [Picea abies (L.) Karst.]. Branches from 17-year-old trees of southern Finnish origin were transferred eight times at 1- to 2-week intervals from October to December 2007 from the field at Punkaharju (61°48'N, 29°20'E) to the greenhouse with forcing conditions (day length 12 h, +20 °C). After seven different durations of forcing, the developmental phase and primordial shoot growth of the buds were analysed at the stereomicroscopic level. Air temperature was recorded hourly throughout the study period. The accumulated chilling unit sum had a significant effect on the temperature sum that was required to attain a certain developmental phase; a higher amount of chilling required a lower amount of forcing. The variation in the rate of development of different buds within each sample branch in relation to the chilling unit and forcing temperature sum was low. Regarding primordial shoot growth, there was also an inverse relation between accumulated chilling and forcing, i.e., a higher accumulated chilling unit sum before forcing required a lower temperature sum to initiate primordial shoot growth and resulted in a stronger effect of accumulated forcing. A second-order regression model with an interaction of chilling and forcing explained the variation of primordial shoot growth with high precision (R(2) = 0.88). However, further studies are required to determine the final parameter values to be used in phenological modelling.

Novel additive manufactured scaffolds for tissue engineered trachea research.

CONCLUSIONS: This study demonstrates proof of concept for controlled manufacturing methods that utilize novel tailored biopolymers (3D photocuring technology) or conventional bioresorbable polymers (fused deposition modeling, FDM) for macroscopic and microscopic geometry control. The manufactured scaffolds could be suitable for tissue engineering research. OBJECTIVES: To design novel trachea scaffold prototypes for tissue engineering purposes, and to fabricate them by additive manufacturing. METHODS: A commercial 3D model and CT scans of a middle-aged man were obtained for geometrical observations and measurements of human trachea. Model trachea scaffolds with variable wall thickness, interconnected pores, and various degrees of porosity were designed. Photocurable polycaprolactone (PCL) polymer was used with 3D photocuring technology. Thermoplastic polylactide (PLA) and PCL were used with FDM. Cell cultivations were performed for biocompatibility studies. RESULTS: Scaffolds of various sizes and porosities were successfully produced. Both thermoplastic PLA and PCL and photocurable PCL could be used effectively with additive manufacturing technologies to print high-quality tubular porous biodegradable structures. Optical microscopic and SEM images showed the viability of cells. The cells were growing in multiple layers, and biocompatibility of the structures was shown.

Development and growth of primordial shoots in Norway spruce buds before visible bud burst in relation to time and temperature in the field.

The timing of bud development in ecodormancy is critical for trees in boreal and temperate regions with seasonally alternating climates. The development of vegetative buds and the growth of primordial shoots (the primordial shoot ratio) in Norway spruce were followed by the naked eye and at stereo and light microscopic levels in fresh-cut and fixed buds obtained by regular field samplings during the spring of 2007, 2008 and 2009. Buds were collected from 15 randomly selected trees (all 16 years old in 2007) of one southern Finnish half-sib family. The air temperature was recorded hourly throughout the observation period. In 2008 and 2009, initial events in the buds, seen as accumulation of lipid droplets in the cortex area, started in mid-March and were depleted in late April, simultaneously with the early development of vascular tissue and primordial needles. In mid-April 2007, however, the development of the buds was at least 10 days ahead as a result of warm spells in March and early April. Variation in the timing of different developmental phases within and among the sample trees was negligible. There was no clear one-to-one correspondence between the externally visible and the internal development of the buds. The dependence of the primordial shoot ratio on different types of temperature sum was studied by means of regression analysis. High coefficients of determination (R(2) ≈ 95%) were attained with several combinations of the starting time (beginning of the year/vernal equinox), the threshold value (from -3 to +5 °C), and the time step (hour/day) used in the temperature summation, i.e., the prediction power of the primordial shoot ratio models turned out to be high, but the parameter estimate values were not unambiguous. According to our results, temperature sums describe the growth of the primordial shoot inside the bud before bud burst. Thus, the results provide a realistic interpretation for the present phenological models of bud development that are based on temperature sums and external observations of bud burst only, and they also provide new tools for improving the models.

Anatomy and morphology in developing vegetative buds on detached Norway spruce branches in controlled conditions before bud burst.

We studied the light and stereomicroscopic structure of developing vegetative buds from a 16-year-old Norway spruce [Picea abies (L.) Karst.] of southern Finnish origin in relation to temperature sum and to externally visible changes in the buds before and during bud burst in forcing conditions. Branches were collected on 17 January and transferred to the greenhouse where they were first subjected to preforcing conditions (darkness, +4 degrees C) for 7 days and then to the forcing conditions (day length 12 h, +20 degrees C). Buds were sampled 20 times between 17 January and 13 February. Air temperature was recorded hourly throughout the study period. The first microscopic change was a temporary increase in the size and number of lipid droplets before the onset of temperature sum (T > or = +5 degrees C) accumulation. From the 4th to the 9th day under the forcing conditions, tracheids started to develop from the base up to the top of the bud. This was closely synchronized with an observed morphological change in the shape of needle tip from rounded to pointed ones. Development from the first visible change in the bud scales on the 12th forcing day to bud burst took 9 days when the temperature sum was 313 d.d. The temperature sums in our experiment overestimated the requirements of temperature sum for bud development phases measured in the field. Bud development could be divided into four structural phases. The first two phases, i.e., morphological changes in the primary needles, occurred without any externally visible changes in the buds. Thus, these phases have a potential for testing and improving the phenological models, which, up to now, have mainly been based on the bud burst observation by the naked eye.

Effect of bud burst forcing on transcript expression of selected genes in needles of Norway spruce during autumn.

Expression of selected genes in needles of Norway spruce (Picea abies [L.] Karst) was investigated by following their transcription levels during late autumn. Transcription was assessed in mature needles which likely serve as sensor of environmental cues that enable trees in the temperate and boreal regions to change between stages of growth, frost tolerance and bud dormancy. Samples were collected from grafts kept under outdoor conditions and after bud burst forcing in greenhouse at 20 degrees C (12 h darkness) for one week. Transcription was assayed with real-time RT-PCR. During the sampling period, chilling requirement was partially fulfilled, and time to bud burst after forcing was decreased. Of the 27 transcripts studied, expression of 16 was significantly affected either by forcing, sampling time, or interaction between them. PaSAP, PaACP, PaSGS3, PaWRKY, PaDIR9, PaCCCH and dehydrin genes responded drastically to forcing temperatures at all sampling points, showing no correlation with readiness for bud burst. Expression patterns of some vernalization pathway gene homologs PaVIN3, and also of PaMDC, PaLOV1 and PaDAL3 had a clear opposite trends between forcing and outdoor conditions, which could imply their role in chilling accumulation and bud burst regulation/cold acclimation. These genes could constitute putative candidates for further detailed study, whose regulation in needles may be involved in preparation towards bud burst and chilling accumulation sensing.

Dehydrins expression related to timing of bud burst in Norway spruce.

Cold deacclimation and preparation to flushing likely requires rehydration of meristems. Therefore, water stress related genes, such as dehydrins (DHN), might play an important role in providing protection during winter dormancy, deacclimation and bud burst timing processes. Here we report the sequence analysis of several Norway spruce DHN identified in late and early flushing suppressive subtraction hybridization cDNA libraries and in our Norway spruce EST database. We obtained 15 cDNAs, representing eight genes from three distinct types of DHN, and studied differential expression of these genes before and during bud burst in spring, using qRT-PCR. We found the visible reduction in transcript level of most DHN towards the bud burst, supported by a significant down-regulation of the DHN in needles during experimental induction of bud burst applied at three time points during autumn in Norway spruce grafts. For most of the DHN transcripts, their expression levels in late-flushing spruces were significantly higher than in the early flushing ones at the same calendar dates but were remarkably similar at the same bud developmental stage. From our results we may conclude that the difference between the early and the late families is in timing of the molecular processes leading to bud burst due to differences in their response to the increasing temperature in the spring. They are induced much earlier in the early flushing families.

Effects of seed origin and sowing time on timing of height growth cessation of Betula pendula seedlings.

We studied the effects of seed origin and sowing time on height development and timing of height growth cessation of first-year silver birch (Betula pendula Roth) seedlings in a greenhouse experiment. Seeds of seven origins ranging in latitudes from 58 degrees to 67 degrees N were sown at 1-2-week intervals eight times from May 21 to July 30, 2001. The day/night temperature in the greenhouse was set at 20/10 degrees C, but lighting was natural and day length varied accordingly. Seedling height was measured twice a week. The interaction term between seed origin and sowing date was significant, but the pattern of height development and timing of growth cessation depended systematically on latitude of seed origin and sowing date. As seed origin became increasingly northern, growth cessation began earlier and resulted in shorter growth periods. Later sowing dates delayed growth cessation but also shortened the growth period. Final seedling height systematically decreased with increasingly northern origins and with later sowings. Linear regression analysis predicted timing of growth cessation, night length at growth cessation, length of growth period and final seedling height with high precision when the latitude of seed origin and sowing time were predictor variables. The timing of height growth cessation was determined by the seed origin, night length and developmental stage of the seedlings.

Effects of photoperiod and temperature on the timing of bud burst in Norway spruce (Picea abies).

We examined the effects of several photoperiod and temperature regimes imposed during the winter-spring period on the timing of bud burst in rooted cuttings of Norway spruce (Picea abies (L.) Karst.) grown in a greenhouse in Finland. The treatments were initiated in November and December after the cuttings had been exposed to natural chilling and freezing events. Irrespective of the treatments applied, time to bud burst decreased with increased duration of previous exposure to natural chilling and freezing events. Fluctuating day/night temperatures and continuous lengthening of the photoperiod hastened bud burst. Shortening the photoperiod delayed bud burst, suggesting that little or no ontogenetic development toward bud burst takes place during mild periods before the winter solstice. In the case of climatic warming, this phenomenon may prevent the premature onset of growth that has been predicted by computer simulations with models that only consider temperature regulation of bud burst.