High-content drug screening with engineered musculoskeletal tissues

Abstract

Tissue engineering for in vitro drug-screening applications based on tissue function is an active area of translational research. Compared to targeted high-throughput drug-screening methods that rapidly analyze hundreds of thousands of compounds affecting a single biochemical reaction or gene expression, high-content screening (HCS) with engineered tissues is more complex and based on the cumulative positive and negative effects of a compound on the multiple pathways altering tissue function. It may therefore serve as better predictor of in vivo activity and serve as a bridge between high-throughput drug screening and in vivo animal studies. In the case of the musculoskeletal system, tissue function includes determining improvements in the mechanical properties of bone, tendon, cartilage, and, for skeletal muscle, contractile properties such as rate of contraction/relaxation, force generation, fatigability, and recovery from fatigue. HCS of compound banks with engineered tissues requires miniature musculoskeletal organs as well as automated functional testing. The resulting technologies should be rapid, cost effective, and reduce the number of small animals required for follow-on in vivo studies. Identification of compounds that improve the repair/regeneration of damaged tissues in vivo would have extensive clinical applications for treating musculoskeletal disorders.

FIG. 1.

High-throughput drug screening versus high-content drug screening of bioactive compounds.

FIG. 2.

Hydrogel tissue construct (HTC) mechanics. (A) Schematic of the force measurement device. a, Linear actuator moved by a servo motor (±70 μm accuracy); b, isometric force transducer; c, force probe; d, tissue chamber; e, warming plate (37°C) connected to a water circulation bath. The device can automatically indent four samples in one row, and the operator manually translates the mold to the next row. A computer records the force response measured by the isometric transducer and regulates the speed of indentation. (B) Tissue chamber for producing and testing engineered tissues. (C) A force probe approaching an HTC formed between two stainless steel bars. (D) The probe indents the HTC vertically and stretches it longitudinally. (E) The typical size of HTCs was approximately 4 × 4 × 0.8 mm (length × width × thickness), and they are formed in 8 × 8 mm (opening) square wells in the tissue chamber. Reprinted with permission from Ref.

FIG. 3.

Tissue engineering miniature bioartificial muscles (mBAMs) on μposts in a 96-microwell plate. Posts are molded from flexible polydimethylsiloxane 4 mm apart and 7 mm high in 7-mm-diameter wells. mBAM shown is day 4–5 postcasting in the 7-mm-diameter well as described in the text. A 7–8-day postcasting mBAM was stained for sarcomeric tropomyosin and shows well-aligned myofibers.

FIG. 4.

Force measurement with flexible μposts. (A) Mechanical model correlating μpost displacement (δ) with force (F). (B) Section of 96-microwell plate with mBAMs attached to flexible posts. (C) Time course for active force development. mBAMs were electrically stimulated every 2–3 days, μpost deflection was measured optically, and active force generation was calculated. Results are means + SE (standard error) from four separate experiments with n = 8–18 per time point. Error bars are smaller than symbols where not seen. (D) Anabolic effect of insulin-like growth factor-1 (IGF-1) on active force. mBAMs were allowed to differentiate for 6–7 days postcasting and then insulin-like growth factor-1 (100 ng/mL) added to the wells. Each point is the mean + SE; n = 6 mBAMs per group. Adapted from Refs.^,

FIG. 5.

(A) Semiautomated versus. (B) automated high-content screening with engineered tissues.