Kyle D. Jadin

Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels

We have fabricated a hepatic tissue construct using a multilayer photopatterning platform for embedding cells in hydrogels of complex architecture. We first explored the potential of established hepatocyte culture models to stabilize isolated hepatocytes for pho‐toencapsulation (e.g., double gel, Matrigel, cocultivation with nonparenchymal cells). Using photopolymerizable PEG hydrogels, we then tailored both the chemistry and architecture of the hydrogels to further support hepatocyte survival and Hver‐specific function. Specifically, we incorporated adhesive peptides to ligate key integrins on these adhesion‐dependent cells. To identify the appropriate peptides for incorporation, the integrin expression of cultured hepatocytes was monitored by flow cytometry and their functional role in cell adhesion was assessed on full‐length extracellular matrix (ECM) molecules and their adhesive peptide domains. In addition, we modified the hydrogel architecture to minimize barriers to nutrient transport for these highly metabolic cells. Viability of encapsulated cells was improved in photopatterned hydrogels with structural features of 500 μm in width over unpatterned, bulk hydrogels. Based on these findings, we fabricated a multilayer photopatterned PEG hydrogel structure containing the adhesive RGD peptide sequence to ligate the α5β1 integrin of cocultured hepatocytes. Three‐dimensional photopatterned constructs were visualized by digital volumetric imaging and cultured in a continuous flow bioreactor for 12 d where they performed favorably in comparison to unpatterned, unper‐fused constructs. These studies will have impact in the field of liver biology as well as provide enabling tools for tissue engineering of other organs.—Liu Tsang, V., Chen, A. A., Cho, L. M., Jadin, K. D., Sah, R. L., DeLong, S., West, J. L., Bhatia, S. N. Fabrication of 3D hepatic tissues by additive photopatterning of cellular hydrogels. FASEB J. 21, 790–801 (2007)

Chondrocyte viability after intra-articular calcaneal fractures in humans

Background: Chondral damage from the impact of injury may contribute to the high incidence of post-traumatic arthritis after calcaneal fractures, but this has yet to be proven. We sought to study the effect of intra-articular calcaneal fractures on chondrocyte viability and to correlate these effects with injury severity, time from injury to surgery, and patient age and co-morbidities. Methods: Irreducible osteochondral fragments from 12 patients undergoing operative treatment for intra-articular calcaneal fractures were analyzed. Control cartilage was obtained from four tissue donors who died of unrelated causes. The cartilage was assessed for chondrocyte viability through the full thickness of tissue using a Live/Dead assay followed by laser scanning confocal microscopy. Patient demographics including injury classification and severity, time from injury to surgery, and patient age were recorded. Results: Chondrocyte viability from fracture patients averaged 72.8% ± 12.9% (range 53% to 95%), which was significantly lower than the 94.8% ± 1.5% viability observed in the control specimens (p = 0.005). Chondrocyte viability declined with higher energy injuries (p = 0.13), time from injury to surgery (p = 0.07), and increasing patient age (p = 0.07). However, none of these factors reached a level of statistical significance. Conclusions: A significant decline in chondrocyte viability occurs after intra-articular fractures of the calcaneus. This may contribute to the development of post-traumatic arthritis.

Effect of 3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic cells.

Various biomaterial scaffolds have been investigated for cartilage tissue engineering, although little attention has been paid to the effect of scaffold microstructure on tissue growth. Non-woven, fibrous, bioabsorbable scaffolds constructed from a copolymer of glycolide and trimethylene carbonate with varying levels of porosity and pore size were seeded with mesenchymal stroma cells with a chondrogenic lineage. Scaffolds and media were evaluated for both cell and extracellular matrix organization and content after up to 28 days of culture in a spinner flask. Analysis of DNA and glycosaminoglycan contents showed that the most porous of the three scaffold types, with a porosity of 81% and a porometry determined mean flow pore diameter of 54 microm, supported the most rapid proliferation of cells and accumulation of extracellular matrix. Analysis of the high porosity scaffold system, using Western Blot and immunohistochemistry confirmed the presence of collagen type II and absence of collagen type I, and demonstrated cells with a chondrocyte morphology with aggrecan and collagen II accumulation attached to the scaffolds. It was concluded that the 3D-microstructural characteristics of the scaffold (interconnecting porosity and pore size) play an important role in proliferation and phenotype of chondrogenic cells and accumulation of extracellular matrix molecules.

Semi-Permeable Membrane Retention of Synovial Fluid Lubricants Hyaluronan and Proteoglycan 4 for a Biomimetic Bioreactor

Synovial fluid (SF) contains lubricant macromolecules, hyaluronan (HA), and proteoglycan 4 (PRG4). The synovium not only contributes lubricants to SF through secretion by synoviocyte lining cells, but also concentrates lubricants in SF due to its semi‐permeable nature. A membrane that recapitulates these synovium functions may be useful in a bioreactor system for generating a bioengineered fluid (BF) similar to native SF. The objectives were to analyze expanded polytetrafluoroethylene membranes with pore sizes of 50 nm, 90 nm, 170 nm, and 3 µm in terms of (1) HA and PRG4 secretion rates by adherent synoviocytes, and (2) the extent of HA and PRG4 retention with or without synoviocytes adherent on the membrane. Experiment 1: Synoviocytes were cultured on tissue culture (TC) plastic or membranes ± IL‐1β + TGF‐β1 + TNF‐α, a cytokine combination that stimulates lubricant synthesis. HA and PRG4 secretion rates were assessed by analysis of medium. Experiment 2: Bioreactors were fabricated to provide a BF compartment enclosed by membranes ± adherent synoviocytes, and an external compartment of nutrient fluid (NF). A solution with HA (1 mg/mL, MW ranging from 30 to 4,000 kDa) or PRG4 (50 µg/mL) was added to the BF compartment, and HA and PRG4 loss into the NF compartment after 2, 8, and 24 h was determined. Lubricant loss kinetics were analyzed to estimate membrane permeability. Experiment 1: Cytokine‐regulated HA and PRG4 secretion rates on membranes were comparable to those on TC plastic. Experiment 2: Transport of HA and PRG4 across membranes was lowest with 50 nm membranes and highest with 3 µm membranes, and transport of high MW HA was decreased by adherent synoviocytes (for 50 and 90 nm membranes). The permeability to HA mixtures for 50 nm membranes was ∼20 × 10−8 cm/s (− cells) and ∼5 × 10−8 cm/s (+ cells), for 90 nm membranes was ∼35 × 10−8 cm/s (− cells) and ∼19 × 10−8 cm/s (+ cells), for 170 nm membranes was ∼74 × 10−8 cm/s (± cells), and for 3 µm membranes was ∼139 × 10−8 cm/s (± cells). The permeability of 450 kDa HA was ∼40× lower than that of 30 kDa HA for 50 nm membranes, but only ∼2.5× lower for 3 µm membranes. The permeability of 4,000 kDa HA was ∼250× lower than that of 30 kDa HA for 50 nm membranes, but only ∼4× lower for 3 µm membranes. The permeability for PRG4 was ∼4 × 10−8 cm/s for 50 nm membranes, ∼48 × 10−8 cm/s for 90 nm membranes, ∼144 × 10−8 cm/s for 170 nm membranes, and ∼336 × 10−8 cm/s for 3 µm membranes. The associated loss across membranes after 24 h ranged from 3% to 92% for HA, and from 3% to 93% for PRG4. These results suggest that semi‐permeable membranes may be used in a bioreactor system to modulate lubricant retention in a bioengineered SF, and that synoviocytes adherent on the membranes may serve as both a lubricant source and a barrier for lubricant transport. Biotechnol. Bioeng. 2010; 106: 149–160.

Characterization of Lesions of the Talus and Description of Tram-Track Lesions:

Background: The ankle joint, although not prone to primary osteoarthritis (OA), is known to be susceptible to secondary OA as a result of sports injuries and other trauma. Unlike the knee joint, a thorough investigation of talar cartilage lesions has not been previously reported. Methods: One hundred and five human tali from 67 donors were used to determine the type and location of the most common lesions through gross examination, radiography, diffraction enhanced imaging, and histology. “tram-track lesions” also are described. Results: The most anterior and posterior regions of the talar dome, along with the medial and lateral borders of the dome were most affected by cartilage degeneration. These are regions that appear to be most subjected to frictional forces from their articulating counterparts during high stress activities that move articulating surfaces slightly out of congruence with each other. One particularly striking cartilage degeneration pattern that, to our knowledge, has not been described histologically, is the tram-track lesion. These lesions displayed a longitudinally oriented groove in the cartilage, running from anterior to posterior, in which the deepest portion of the groove was located in the middle of the anterior-posterior axis of the talus. Several of these cartilage grooves had a bony ridge beneath, mirroring the cartilage groove whereas others did not. This suggests that the cartilage groove develops before the interruption of the tidemark (border between the calcified and uncalcified cartilage). In specimens for which the articulating tibial articular surface was available, (17) it was found that the tibia displayed small osteophytes on the anterior articular margin that exactly corresponded to their articulation with the talar cartilage grooves as the ankle articulated through plantar flexion and dorsiflexion. Conclusion: This study provides an in-depth histologic and gross anatomic look at the most common lesions of the talus of the ankle joint. In particular, the “tram-track” lesion was shown to be a consequence of its tibial articulation and to include both subchondral bone and articular cartilage changes.