Michele Marcolongo

Characterization of cell viability during bioprinting processes

Bioprinting is an emerging technology in the field of tissue engineering and regenerative medicine. The process consists of simultaneous deposition of cells, biomaterial and/or growth factors under pressure through a micro-scale nozzle. Cell viability can be controlled by varying the parameters like pressure and nozzle diameter. The process itself can be a very useful tool for evaluating an in vitro cell injury model. It is essential to understand the cell responses to process-induced mechanical disturbances because they alter cell morphology and function. We carried out analysis and quantification of the degree of cell injury induced by bioprinting process. A parametric study with different process parameters was conducted to analyze and quantify cell injury as well as to optimize the parameters for printing viable cells. A phenomenological model was developed correlating the percentage of live, apoptotic and necrotic cells to the process parameters. This study incorporates an analytical formulation to predict the cell viability through the system as a function of the maximum shear stress in the system. The study shows that dispensing pressure has a more significant effect on cell viability than the nozzle diameter. The percentage of live cells is reduced significantly (by 38.75%) when constructs are printed at 40 psi compared to those printed at 5 psi.

Degradation of mechanical properties of UHMWPE acetabular liners following long-term implantation☆

We tested the hypothesis that the mechanical and chemical behavior of gamma radiation-sterilized ultrahigh-molecular-weight polyethylene (UHMWPE) changes after implantation. Relationships between the mechanical behavior and oxidation index were explored in a cohort of 16 consecutive traceable Hexloc acetabular components (Biomet, Warsaw, IN) that were machined from extruded, stearate-containing UHMWPE and gamma sterilized in air. Shelf aging time (average, 0.4 years) and implantation time (average, 11.5 years) were determined for all 16 inserts. The retrieved liners exhibited significant mechanical degradation, which was most severe in the unloaded surface regions. Analysis of the Fourier transform infrared spectroscopy data revealed a significant association between the oxidation index and mechanical degradation of the UHMWPE. The results of this study strongly support the hypothesis that the degradation of mechanical properties for the liners occurred during implantation.

In vivo degradation of polyethylene liners after gamma sterilization in air.

BACKGROUND Ultra-high molecular weight polyethylene degrades during storage in air following gamma sterilization, but the extent of in vivo degradation remains unclear. The purpose of this study was to quantify the extent to which the mechanical properties and oxidation of conventional polyethylene acetabular liners treated with gamma sterilization in air change in vivo. METHODS Fourteen modular cementless acetabular liners were revised at an average of 10.3 years (range, 5.9 to 13.5 years) after implantation. All liners, which had been machined from GUR 415 resin, had been gamma-sterilized in air; the average shelf life was 0.3 year (range, 0.0 to 0.8 year). After removal, the components were expeditiously frozen to minimize ex vivo changes to the polyethylene prior to characterization. The average duration between freezing and testing was 0.6 year. Mechanical properties and oxidation were measured with use of the small-punch test and Fourier transform infrared spectroscopy, respectively, in the loaded and unloaded regions of the liners. RESULTS There was substantial regional variation in the mechanical properties and oxidation of the retrieved liners. The ultimate load was observed to vary by >90% near the surface. On the average, the rim and the unloaded bearing showed evidence of severe oxidation near the surface after long-term in vivo aging, but these trends were not typically observed on the loaded bearing surface or near the backside of the liners. CONCLUSIONS The mechanical properties of polyethylene that has been gamma-sterilized in air may decrease substantially in vivo, depending on the location in the liner. The most severe oxidation was observed at the rim, suggesting that the femoral head inhibits access of oxygen-containing body fluids to the bearing surface. This is perhaps why in vivo oxidation has not been associated with clinical performance to date.

Synthesis and recovery characteristics of branched and grafted PNIPAAm–PEG hydrogels for the development of an injectable load-bearing nucleus pulposus replacement

A family of injectable poly(N-isopropyl acrylamide) (PNIPAAm) copolymer hydrogels has been fabricated in order to tune mechanical properties to support load-bearing function and dimensional recovery for possible use as load-bearing medical devices, such as a nucleus pulposus replacement for the intervertebral disc. PNIPAAm-polyethylene glycol (PEG) copolymers were synthesized with varying hydrophilic PEG concentrations as grafted or branched structures to enhance dimensional recovery of the materials. Polymerizations were confirmed with attenuated total reflectance-Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy studies. Incorporation of PEG was effective in raising water content of pure PNIPAAm hydrogels (29.3% water for pure PNIPAAm vs. 47.7% for PEG branches and 39.5% for PEG grafts). PNIPAAm with 7% grafted as well as 7% branched PEG had significantly reduced compressive modulus compared to that of pure PNIPAAm. Initially recovered compressive strain was significantly increased for 7% PEG branches after pre-testing immersion in PBS for up to 33 days, while 7% PEG grafts decreased this value. Sample height recovery for pure PNIPAAm was limited to 31.6%, while PNIPAAm with 7% branches was increased to 71.3%. When mechanically tested samples were allowed to recover without load over 30 min, each composition was able to significantly recover height, indicating that the time to recovery is slower than the unloading rates typically used in testing. While the incorporation of hydrophilic PEG was expected to alter the mechanical behavior of the hydrogels, only the branched form was able to significantly enhance dimensional recovery.

Nucleus Implant Parameters Significantly Change the Compressive Stiffness of the Human Lumbar Intervertebral Disc

Nucleus replacement by a synthetic material is a recent trend for treatment of lower back pain. Hydrogel nucleus implants were prepared with variations in implant modulus, height, and diameter Human lumbar intervertebral discs (IVDs) were tested in compression for intact, denucleated, and implanted condition. Implantation of nucleus implants with different material and geometric parameters into a denucleated IVD significantly altered the IVD compressive stiffness. Variations in the nucleus implant parameters significantly change the compressive stiffness of the human lumbar IVD. Implant geometrical variations were more effective than those of implant modulus variations in the range examined.

The effect of protein-free versus protein-containing medium on the mechanical properties and uptake of ions of PVA/PVP hydrogels.

The effect of two simulated biological environments (protein-free and protein-containing) on ion uptake and physical properties of PVA/PVP hydrogels were explored in this work. It was found that over the immersion period in both media, wet mass of the hydrogels decreased and compressive moduli increased, likely due to increased polymer content with water loss as the hydrogels equilibrated with water. These changes were independent of polymer content and immersion medium. However, dry mass of the hydrogels increased dramatically when immersed in protein-free medium, changing only moderately in protein-containing medium. The increase in dry mass was attributed to ion uptake from immersion medium, as confirmed by EDXA. We postulate that differences between ion uptake in protein-free versus protein-containing medium is likely the result of serum proteins in the proteincontaining medium adsorbing to the surface, inhibiting transport of ions into the hydrogel.

Effects of aging and degeneration on the human intervertebral disc during the diurnal cycle: A finite element study

A significant biochemical change that takes place in intervertebral disc degeneration is the loss of proteoglycans in the nucleus pulposus. Proteoglycans attract fluid, which works to reduce mechanical stresses in the solid matrix of the nucleus and provide a hydrostatic pressure to the annulus fibrosus, whose fibrous nature accommodates this stress. Our goals are to develop an osmo‐poroelastic finite element model to study the relationship between proteoglycan content and the stress distribution within the disc and to analyze the effects of degeneration on the discs diurnal mechanical response. Stress in the annulus increased with degeneration from ∼0.2 to 0.4 MPa, and an increase occurred in the center of the nucleus from 1.2 to 1.6 MPa. The osmotic pressure in the central nucleus region decreased the most with degeneration, from ∼0.42 to ∼0.1 MPa in a severely dehydrated disc. A 3% decrease in diurnal fluid lost with degeneration equated to ∼21% decrease in fluid exchange, and hence a decrease in nutrients that require convection to enter the disc. We quantified the increases in internal stresses in the nucleus and annulus throughout the various stages of degeneration, suggesting that these changes lead to further remodeling of the tissue.

23Na TQF NMR imaging for the study of spinal disc tissue

A method for acquiring triple quantum filtered (TQF) (23)Na NMR images is proposed that takes advantage of the differences in transverse relaxation rates of sodium to achieve positive intensity, PI, NMR signal. This PITQF imaging sequence has been used to obtain spatially resolved one-dimensional images as a function of the TQF creation time, tau, for two human spinal disc samples. From the images the different parts of the tissue, nucleus pulposus and annulus fibrosus, can be clearly distinguished based on their signal intensity and creation time profiles. These results establish the feasibility of (23)Na TQF imaging and demonstrate that this method should be applicable for studying human disc tissues as well as spinal disc degeneration.

Comparison of periprosthetic tissue digestion methods for ultra-high molecular weight polyethylene wear debris extraction.

There is considerable interest in characterization of wear debris from polyethylene (UHMWPE) bearing components used in total joint replacement. To isolate UHMWPE wear debris, tissue samples must be excised from regions adjacent to revised UHMWPE implant components, followed by exposure to one of many available tissue digestion methods. Numerous studies demonstrate successful digestion, but the relative efficiency of each method is not clear. The purpose of this study was to evaluate a variety of conditions for tissue digestion to provide a quantitative comparison of methods. Porcine and human hip tissues were exposed for 24 h to basic, acidic or enzymatic agents, filtered and digestion efficiency calculated based on the percentage of initial to final tissue weight. Of the conditions tested, 5 M NaOH, 5 M KOH, 15 M KOH or 15.8 M HNO(3) yielded the most complete porcine hip tissue digestion (<1% residual tissue weight; p < 0.05). Proteinase K and Liberase Blendzyme 3 did not effectively digest tissue in a 24 h period. Similar to results from the porcine dataset, human tissues digestion was most efficient using 5 M NaOH, 5 M KOH or 15.8 M HNO(3) (<1% residual tissue weight; p < 0.05). To verify that particle surface modifications did not occur after prolonged reagent exposure, GUR415 and Ceridust 3715 particles were immersed in each solution for 24 h. Overall, this study provides a framework for thorough and efficient digestive methods for UHMWPE wear debris extraction.

2H double quantum filtered (DQF) NMR spectroscopy of the nucleus pulposus tissues of the intervertebral disc

Deuterium (2H) double‐quantum filtered (DQF) NMR spectroscopy of nucleus pulposus (NP) tissues from human intervertebral discs is reported. The DQF spectral intensities, DQ build‐up rates, and DQF‐detected rotating‐frame spin‐lattice relaxation times are sensitive to the degree of hydration of the NP tissue, and display a monotonous correlation with age between 15 and 80 years. The implications of this work are that the changes in water dynamics as detected via DQF NMR spectroscopy may be used as a probe of tissue degeneration in NP, particularly in the early stages of degeneration to which most standard NMR methods are not sensitive. Magn Reson Med 57:990–999, 2007.