Improved Chondrogenic Potential and Proteomic Phenotype of Porcine Chondrocytes Grown in Optimized Culture Conditions.

For successful cartilage tissue engineering, the ability to generate a high number of chondrocytes in vitro while avoiding terminal differentiation or de-differentiation is critical. The ability to accomplish this by using the abundant and easily sampled costal cartilage could provide a practical alternative to the use of articular cartilage and mesenchymal stem cells. Chondrocytes isolated from pig costal cartilage were expanded in either serum-free medium with FGF2 (SFM) or fetal bovine serum-containing medium (SCM), under either high (21%) or low (5%) oxygen conditions. Overall, chondrocytes cultured in SFM and low oxygen (Low-SFM) demonstrated the highest cell growth rate (p < 0.05). The effect of passage number on the differentiation status of the chondrocytes was analyzed by alkaline phosphatase (AP) staining and real-time PCR for known chondrocyte quality markers. AP staining indicated that chondrocytes grown in SCM had a higher proportion of terminally differentiated (hypertrophic) chondrocytes (p < 0.05). At the mRNA level, expression ratios of ACAN/VCAN and COL2/COL1 were significantly higher (p < 0.05) in cells expanded in Low-SFM, indicating reduced de-differentiation. In vitro re-differentiation capacity was assessed after a 6-week induction, and chondrocytes grown in Low-SFM showed similar expression ratios of COL2/COL1 and ACAN/VCAN to native cartilage. Proteomic analysis of in vitro produced cartilage indicated that the Low-SFM condition most closely matched the proteomic profile of native costal and articular cartilage. In conclusion, Low-SFM culture conditions resulted in improved cell growth rates, reduced levels of de-differentiation during expansion, greater ability to re-differentiate into cartilage on induction, and an improved proteomic profile that resembles that of in vivo cartilage.

Bioprinting: an assessment based on manufacturing readiness levels.

Over the last decade, bioprinting has emerged as a promising technology in the fields of tissue engineering and regenerative medicine. With recent advances in additive manufacturing, bioprinting is poised to provide patient-specific therapies and new approaches for tissue and organ studies, drug discoveries and even food manufacturing. Manufacturing Readiness Level (MRL) is a method that has been applied to assess manufacturing maturity and to identify risks and gaps in technology-manufacturing transitions. Technology Readiness Level (TRL) is used to evaluate the maturity of a technology. This paper reviews recent advances in bioprinting following the MRL scheme and addresses corresponding MRL levels of engineering challenges and gaps associated with the translation of bioprinting from lab-bench experiments to ultimate full-scale manufacturing of tissues and organs. According to our step-by-step TRL and MRL assessment, after years of rigorous investigation by the biotechnology community, bioprinting is on the cusp of entering the translational phase where laboratory research practices can be scaled up into manufacturing products specifically designed for individual patients.

Manufacturing road map for tissue engineering and regenerative medicine technologies.

The Regenerative Medicine Foundation Annual Conference held on May 6 and 7, 2014, had a vision of assisting with translating tissue engineering and regenerative medicine (TERM)-based technologies closer to the clinic. This vision was achieved by assembling leaders in the field to cover critical areas. Some of these critical areas included regulatory pathways for regenerative medicine therapies, strategic partnerships, coordination of resources, developing standards for the field, government support, priorities for industry, biobanking, and new technologies. The final day of this conference featured focused sessions on manufacturing, during which expert speakers were invited from industry, government, and academia. The speakers identified and accessed roadblocks plaguing the field where improvements in advanced manufacturing offered many solutions. The manufacturing sessions included (a) product development toward commercialization in regenerative medicine, (b) process challenges to scale up manufacturing in regenerative medicine, and (c) infrastructure needs for manufacturing in regenerative medicine. Subsequent to this, industry was invited to participate in a survey to further elucidate the challenges to translation and scale-up. This perspective article will cover the lessons learned from these manufacturing sessions and early results from the survey. We also outline a road map for developing the manufacturing infrastructure, resources, standards, capabilities, education, training, and workforce development to realize the promise of TERM.

Interdigitated silver-polymer-based antibacterial surface system activated by oligodynamic iontophoresis - an empirical characterization study.

There is a pressing need to control the occurrences of nosocomial infections due to their detrimental effects on patient well-being and the rising treatment costs. To prevent the contact transmission of such infections via health-critical surfaces, a prophylactic surface system that consists of an interdigitated array of oppositely charged silver electrodes with polymer separations and utilizes oligodynamic iontophoresis has been recently developed. This paper presents a systematic study that empirically characterizes the effects of the surface system parameters on its antibacterial efficacy, and validates the system's effectiveness. In the first part of the study, a fractional factorial design of experiments (DOE) was conducted to identify the statistically significant system parameters. The data were used to develop a first-order response surface model to predict the system's antibacterial efficacy based on the input parameters. In the second part of the study, the effectiveness of the surface system was validated by evaluating it against four bacterial species responsible for several nosocomial infections - Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Enterococcus faecalis - alongside non-antibacterial polymer (acrylic) control surfaces. The system demonstrated statistically significant efficacy against all four bacteria. The results indicate that given a constant total effective surface area, the system designed with micro-scale features (minimum feature width: 20 µm) and activated by 15 µA direct current will provide the most effective antibacterial prophylaxis.

Nanomaterials and synergistic low-intensity direct current (LIDC) stimulation technology for orthopedic implantable medical devices.

Nanomaterials play a significant role in biomedical research and applications because of their unique biological, mechanical, and electrical properties. In recent years, they have been utilized to improve the functionality and reliability of a wide range of implantable medical devices ranging from well-established orthopedic residual hardware devices (e.g., hip implants) that can repair defects in skeletal systems to emerging tissue engineering scaffolds that can repair or replace organ functions. This review summarizes the applications and efficacies of these nanomaterials that include synthetic or naturally occurring metals, polymers, ceramics, and composites in orthopedic implants, the largest market segment of implantable medical devices. The importance of synergistic engineering techniques that can augment or enhance the performance of nanomaterial applications in orthopedic implants is also discussed, the focus being on a low-intensity direct electric current (LIDC) stimulation technology to promote the long-term antibacterial efficacy of oligodynamic metal-based surfaces by ionization, while potentially accelerating tissue growth and osseointegration. While many nanomaterials have clearly demonstrated their ability to provide more effective implantable medical surfaces, further decisive investigations are necessary before they can translate into medically safe and commercially viable clinical applications. The article concludes with a discussion about some of the critical impending issues with the application of nanomaterials-based technologies in implantable medical devices, and potential directions to address these.

Micro-scale fabrication and characterization of a silver-polymer-based electrically activated antibacterial surface.

This paper reports the fabrication methodology and characterization results for an electrically activated silver-polymer-based antibacterial surface with primary applications in preventing indirect contact transmission of infections. The surface consists of a micro-scale grating pattern of alternate silver electrodes and SU-8 partitions with a minimum feature size of 20 µm, and activated by an external voltage. In this study, prototype coupons (15 mm × 15 mm) of the antibacterial surface were fabricated on silicon substrates using two sets of lithographies, and analyzed for their physical characteristics using microscopy and surface profilometry. The prototypes were also electrically analyzed to determine their current-voltage characteristics, and hence silver ion (Ag(+)) release concentrations. Finally, they were tested for their antibacterial efficacy against Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) using a newly engineered microbiological testing procedure. The antibacterial efficacy testing results show significant reductions in the number of viable organisms of both the species after 45 min of testing with 15 µA system current. Due to the growing incidences of hospital-acquired infections and rising treatment costs, study and application of such alternative antibacterial systems in critical touch-contact and work surfaces (e.g., door push plates, countertops, medical instrument trays) for healthcare environments has become essential.

Developing an engineered antimicrobial/prophylactic system using electrically activated bactericidal metals.

The increased use of Residual Hardware Devices (RHDs) in medicine combined with antimicrobial resistant-bacteria make it critical to reduce the number of RHD associated osteomyelitic infections. This paper proposes a surface treatment based on ionic emission to create an antibiotic environment that can significantly reduce RHD associated infections. The Kirby-Bauer agar gel diffusion technique was adopted to examine the antimicrobial efficacy of eight metals and their ionic forms against seven microbes commonly associated with osteomyelitis. Silver ions (Ag(+)) showed the most significant bactericidal efficacy. A second set of experiments, designed to identify the best configuration and operational parameters for Ag(+) based RHDs addressed current and ionic concentrations by identifying and optimizing parameters including amperage, cathode and anode length, separation between anode and cathode, and surface charge density. The system demonstrated an unparalleled efficacy. The concept was then implemented during in vitro testing of an antimicrobial hip implant, RHD.