A cell bank paradigm for preclinical evaluation of an analogous cellular product for an allogeneic cell therapy.

Toward the translation of allogeneic cell therapy products, cell banks are needed not only to manufacture the final human product but also during the preclinical evaluation of an animal-based analogous cellular product (ACP). These cell banks need to be established at both the master cell bank (MCB) level and the working cell bank (WCB) level. Inasmuch as most of the development of cell therapy products is at academic centers, it is imperative that academic researchers understand how to establish MCBs and WCBs within an academic environment. To illustrate this process, using articular cartilage as the model, a cell bank for an ACP was developed (MCBs at passage 2, WCBs at passage 5) to produce self-assembled neocartilage for preclinical evaluation (constructs at passage 7). The cell bank system is estimated to be able to produce between 160 000 and 400 000 constructs for each of the six MCBs. Overall, the ACP cell bank yielded constructs that are analogous to the intended human product, which is critical toward conducting preclinical evaluations of the ACP for inclusion in an Investigational New Drug application to the FDA.

Vibrometry as a noncontact alternative to dynamic and viscoelastic mechanical testing in cartilage.

Physiological loading of knee cartilage is highly dynamic and may contribute to the progression of osteoarthritis. Thus, an understanding of cartilage's dynamic mechanical properties is crucial in cartilage research. In this study, vibrometry was used as a fast (2 h), noncontact and novel alternative to the slower (30 h), traditional mechanical and biochemical assays for characterization of cartilage from the condyle, patella, trochlear groove and meniscus. Finite-element models predicted tissue resonant frequencies and bending modes, which strongly correlated with experiments (R2 = 0.93). Vibrometry-based viscoelastic properties significantly correlated with moduli from stress relaxation and creep tests, with correlation strengths reaching up to 0.78. Loss modulus also strongly correlated with glycosoaminoglycan (GAG) content. Dynamic properties measured by vibrometry significantly differed among various knee cartilages, ranging between 6.1 and 56.4 MPa. Interestingly, meniscus viscoelastic properties suggest that contrary to common belief, it may lack shock absorption abilities; instead, condylar hyaline cartilage may be a better shock absorber. These data demonstrate for the first time that vibrometry is a noncontact approach to dynamic mechanical characterization of hyaline and fibrocartilage cartilage with concrete relationships to standard quasi-static mechanical testing and biochemical composition. Thus, with a single tool, vibrometry greatly facilitates meeting multiple regulatory recommendations for mechanical characterization of cartilage replacements.

A Tribological Comparison of Facet Joint, Sacroiliac Joint, and Knee Cartilage in the Yucatan Minipig.

OBJECTIVE: Pathology of the facet and sacroiliac (SI) joints contributes to 15% to 45% and 10% to 27% of lower back pain cases, respectively. Although tissue engineering may offer novel treatment options to patients suffering from cartilage degeneration in these joints, the tribological characteristics of the facet and SI joints have not been studied in either the human or relevant large animal models, which hinders the development of joint-specific cartilage implants. DESIGN: Cartilage was isolated from the knee, cervical facet, thoracic facet, lumbar facet, and SI joints of 6 skeletally mature Yucatan minipigs (Sus scrofa). Tribological characteristics were assessed via coefficient of friction testing, interferometry, and immunohistochemistry for lubricin organization. RESULTS: Compared with the knee, the coefficient of friction was higher by 43% in the cervical facet, 77% in the thoracic facet, 37% in the lumbar facet, and 28% in the SI joint. Likewise, topographical features of the facet and SI joints varied significantly, ranging from a 114% to 384% increase and a 48% to 107% increase in global and local surface roughness measures, respectively, compared with the knee. Additionally, the amount of lubricin in the SI joint was substantially greater than in the knee. Statistical correlations among the various tribological parameters revealed that there was a significant correlation between local roughness and coefficient of friction, but not global roughness or the presence of lubricin. CONCLUSION: These location-specific tribological characteristics of the articular cartilages of the spine will need to be taken into consideration during the development of physiologically relevant, functional, and durable tissue-engineered replacements for these joints.

Knee orthopedics as a template for the temporomandibular joint.

Although the knee joint and temporomandibular joint (TMJ) experience similar incidence of cartilage ailments, the knee orthopedics field has greater funding and more effective end-stage treatment options. Translational research has resulted in the development of tissue-engineered products for knee cartilage repair, but the same is not true for TMJ cartilages. Here, we examine the anatomy and pathology of the joints, compare current treatments and products for cartilage afflictions, and explore ways to accelerate the TMJ field. We examine disparities, such as a 6-fold higher article count and 2,000-fold higher total joint replacement frequency in the knee compared to the TMJ, despite similarities in osteoarthritis incidence. Using knee orthopedics as a template, basic and translational research will drive the development and implementation of clinical products for the TMJ. With more funding opportunities, training programs, and federal guidance, millions of people afflicted with TMJ disorders could benefit from novel, life-changing therapeutics.

Cartilage Assessment Requires a Surface Characterization Protocol: Roughness, Friction, and Function.

The surface of articular cartilage is integral to smooth, low-friction joint articulation. However, the majority of cartilage literature rarely includes measurements of surface characteristics and function. This may, in part, be due to a shortage of or unfamiliarity with fast, nondestructive, and, preferably, noncontact methods that can be applied to large cartilage surfaces for evaluating cartilage surface characteristics. A comprehensive methodology for characterizing cartilage surfaces is useful in determining changes in tissue function, as for example, in cases where the quality of cartilage grafts needs to be assessed. With cartilage storage conditions being an area of ongoing and active research, this study used interferometry and tribology methods as efficient and nondestructive ways of evaluating changes in cartilage surface topography, roughness, and coefficient of friction (CoF) resulting from various storage temperatures and durations. Standard, destructive testing for bulk mechanical and biochemical properties, as well as immunohistochemistry, were also performed. For the first time, interferometry was used to show cartilage topographical anisotropy through an anterior-posterior striated pattern in the same direction as joint articulation. Another novel observation enabled by tribology was frictional anisotropy, illustrated by a 53% increase in CoF in the medial-lateral direction compared to the anterior-posterior direction. Of the storage conditions examined, 37°C, 4°C, -20°C, and -80°C for 1 day, 1 week, and 1 month, a 49% decrease in CoF was observed at 1 week in -80°C. Interestingly, prolonged storage at 37°C resulted in up to an 83% increase in the compressive aggregate modulus by 1 month, with a corresponding increase in the glycosaminoglycan (GAG) bulk content. This study illustrates the differential effects of storage conditions on cartilage: freezing tends to target surface properties, while nonfreezing storage impacts the tissue bulk. These data show that a bulk-only analysis of cartilage function is not sufficient or representative. The nondestructive surface characterization assays described here enable improvement in cartilage functionality assessment by considering both surface and bulk cartilage properties; this methodology may thus provide a new angle to explore in future cartilage research and tissue engineering endeavors.

Molecular and mechanical signals determine morphogenesis of the cerebral hemispheres in the chicken embryo.

During embryonic development, the telecephalon undergoes extensive growth and cleaves into right and left cerebral hemispheres. Although molecular signals have been implicated in this process and linked to congenital abnormalities, few studies have examined the role of mechanical forces. In this study, we quantified morphology, cell proliferation and tissue growth in the forebrain of chicken embryos during Hamburger-Hamilton stages 17-21. By altering embryonic cerebrospinal fluid pressure during development, we found that neuroepithelial growth depends on not only chemical morphogen gradients but also mechanical feedback. Using these data, as well as published information on morphogen activity, we developed a chemomechanical growth law to mathematically describe growth of the neuroepithelium. Finally, we constructed a three-dimensional computational model based on these laws, with all parameters based on experimental data. The resulting model predicts forebrain shapes consistent with observations in normal embryos, as well as observations under chemical or mechanical perturbation. These results suggest that molecular and mechanical signals play important roles in early forebrain morphogenesis and may contribute to the development of congenital malformations.

Reduced embryonic blood flow impacts extracellular matrix deposition in the maturing aorta.

BACKGROUND: Perturbations to embryonic hemodynamics are known to adversely affect cardiovascular development. Vitelline vein ligation (VVL) is a model of reduced placental blood flow used to induce cardiac defects in early chick embryo development. The effect of these hemodynamic interventions on maturing elastic arteries is largely unknown. We hypothesize that hemodynamic changes impact maturation of the dorsal aorta (DA). RESULTS: We examined the effects of VVL on hemodynamic properties well into the maturation process and the corresponding changes in aortic dimensions, wall composition, and gene expression. In chick embryos, we found that DA blood velocity was reduced immediately postsurgery at Hamburger-Hamilton (HH) stage 18 and later at HH36, but not in the interim. Throughout this period, DA diameter adapted to maintain a constant shear stress. At HH36, we found that VVL DAs showed a substantial decrease in elastin and a modest increase in collagen protein content. In VVL DAs, up-regulation of elastic fiber-related genes followed the down-regulation of flow-dependent genes. Together, these suggest the existence of a compensatory mechanism in response to shear-induced delays in maturation. CONCLUSIONS: The DA's response to hemodynamic perturbations invokes coupled mechanisms for shear regulation and matrix maturation, potentially impacting the course of vascular development. Developmental Dynamics 247:914-923, 2018. © 2018 Wiley Periodicals, Inc.