Effects of chondrogenic priming duration on mechanoregulation of engineered cartilage.

Chondrocyte maturation during cartilage development occurs under diverse and dynamic mechanical environments. Mechanical stimulation through bioreactor culture may mimic these conditions to direct cartilage tissue engineering in vitro. Mechanical cues can promote chondrocyte homeostasis or hypertrophy and mineralization, depending potentially on the timing of load application. Here, we tested the effects of chondrogenic priming duration on the response of engineered human cartilage constructs to dynamic mechanical compression. We cultured human bone marrow stromal cells (hMSCs) in fibrin hydrogels under chondrogenic priming conditions for periods of 0, 2, 4, or 6 weeks prior to two weeks of either static culture or dynamic compression. We measured construct mechanical properties, cartilage matrix composition, and gene expression. Dynamic compression increased the equilibrium and dynamic modulus of the engineered tissue, depending on the duration of chondrogenic priming. For priming times of 2 weeks or greater, dynamic compression enhanced COL2A1 and AGGRECAN mRNA expression at the end of the loading period, but did not alter total collagen or glycosaminoglycan matrix deposition. Load initiation at priming times of 4 weeks or less repressed transient osteogenic signaling (RUNX2, OPN) and expression of CYR61, a YAP/TAZ-TEAD-target gene. No suppression of osteogenic gene expression was observed if loading was initiated after 6 weeks of in vitro priming, when mechanical stimulation was observed to increase the expression of type X collagen. Taken together, these data demonstrate that the duration of in vitro chondrogenic priming regulates the cell response to dynamic mechanical compression and suggests that early loading may preserve chondrocyte homeostasis while delayed loading may support cartilage maturation.

Recapitulating bone development through engineered mesenchymal condensations and mechanical cues for tissue regeneration.

Large bone defects cannot form a callus and exhibit high complication rates even with the best treatment strategies available. Tissue engineering approaches often use scaffolds designed to match the properties of mature bone. However, natural fracture healing is most efficient when it recapitulates development, forming bone via a cartilage intermediate (endochondral ossification). Because mechanical forces are critical for proper endochondral bone development and fracture repair, we hypothesized that recapitulating developmental mechanical forces would be essential for large bone defect regeneration in rats. Here, we engineered mesenchymal condensations that mimic the cellular organization and lineage progression of the early limb bud in response to local transforming growth factor-ß1 presentation from incorporated gelatin microspheres. We then controlled mechanical loading in vivo by dynamically tuning fixator compliance. Mechanical loading enhanced mesenchymal condensation-induced endochondral bone formation in vivo, restoring functional bone properties when load initiation was delayed to week 4 after defect formation. Live cell transplantation produced zonal human cartilage and primary spongiosa mimetic of the native growth plate, whereas condensation devitalization before transplantation abrogated bone formation. Mechanical loading induced regeneration comparable to high-dose bone morphogenetic protein-2 delivery, but without heterotopic bone formation and with order-of-magnitude greater mechanosensitivity. In vitro, mechanical loading promoted chondrogenesis and up-regulated pericellular matrix deposition and angiogenic gene expression. In vivo, mechanical loading regulated cartilage formation and neovascular invasion, dependent on load timing. This study establishes mechanical cues as key regulators of endochondral bone defect regeneration and provides a paradigm for recapitulating developmental programs for tissue engineering.

Influence of structural load-bearing scaffolds on mechanical load- and BMP-2-mediated bone regeneration.

A common design constraint in functional tissue engineering is that scaffolds intended for use in load-bearing sites possess similar mechanical properties to the replaced tissue. Here, we tested the hypothesis that in vivo loading would enhance bone morphogenetic protein-2 (BMP-2)-mediated bone regeneration in the presence of a load-bearing PLDL scaffold, whose pores and central core were filled with BMP-2-releasing alginate hydrogel. First, we evaluated the effects of in vivo mechanical loading on bone regeneration in the structural scaffolds. Second, we compared scaffold-mediated bone regeneration, independent of mechanical loading, with alginate hydrogel constructs, without the structural scaffold, that have been shown previously to facilitate in vivo mechanical stimulation of bone formation. Contrary to our hypothesis, mechanical loading had no effect on bone formation, distribution, or biomechanical properties in structural scaffolds. Independent of loading, the structural scaffolds reduced bone formation compared to non-structural alginate, particularly in regions in which the scaffold was concentrated, resulting in impaired functional regeneration. This is attributable to a combination of stress shielding by the scaffold and inhibition of cellular infiltration and tissue ingrowth. Collectively, these data question the necessity of scaffold similarity to mature tissue at the time of implantation and emphasize development of an environment conducive to cellular activation of matrix production and ultimate functional regeneration.

Net shape fabrication of calcium phosphate scaffolds with multiple material domains.

Calcium phosphate (CaP) materials have been proven to be efficacious as bone scaffold materials, but are difficult to fabricate into complex architectures because of the high processing temperatures required. In contrast, polymeric materials are easily formed into scaffolds with near-net-shape forms of patient-specific defects and with domains of different materials; however, they have reduced load-bearing capacity compared to CaPs. To preserve the merits of CaP scaffolds and enable advanced scaffold manufacturing, this manuscript describes an additive manufacturing process that is coupled with a mold support for overhanging features; we demonstrate that this process enables the fabrication of CaP scaffolds that have both complex, near-net-shape contours and distinct domains with different microstructures. First, we use a set of canonical structures to study the manufacture of complex contours and distinct regions of different material domains within a mold. We then apply these capabilities to the fabrication of a scaffold that is designed for a 5 cm orbital socket defect. This scaffold has complex external contours, interconnected porosity on the order of 300 µm throughout, and two distinct domains of different material microstructures.

Microcomputed tomography: approaches and applications in bioengineering.

Microcomputed tomography (microCT) has become a standard and essential tool for quantifying structure-function relationships, disease progression, and regeneration in preclinical models and has facilitated numerous scientific and bioengineering advancements over the past 30 years. In this article, we recount the early events that led to the initial development of microCT and review microCT approaches for quantitative evaluation of bone, cartilage, and cardiovascular structures, with applications in fundamental structure-function analysis, disease, tissue engineering, and numerical modeling. Finally, we address several next-generation approaches under active investigation to improve spatial resolution, acquisition time, tissue contrast, radiation dose, and functional and molecular information.