Polylactide (PLA) is the most widely utilized biopolymer in medicine. However, chronic inflammation and excessive fibrosis resulting from its degradation remain significant obstacles to extended clinical use. Immune cell activation has been correlated to the acidity of breakdown products, yet methods to neutralize the pH have not significantly reduced adverse responses. Using a bioenergetic model, we observed delayed cellular changes that were not apparent in the short-term. Amorphous and semi-crystalline PLA degradation products, including monomeric L-lactic acid, mechanistically remodel metabolism in cells leading to a reactive immune microenvironment characterized by elevated proinflammatory cytokines. Selective inhibition of metabolic reprogramming and altered bioenergetics both reduce these undesirable high cytokine levels and stimulate anti-inflammatory signals. Our results present a new biocompatibility paradigm by identifying metabolism as a target for immunomodulation to increase tolerance to biomaterials, ensuring safe clinical application of PLA-based implants for soft- and hard-tissue regeneration, and advancing nanomedicine and drug delivery.

Extracellular vesicles (EVs) are cell-derived nanostructures that mediate intercellular communication by delivering complex signals in normal tissues and cancer. The cellular coordination required for tumor development and maintenance is mediated, in part, through EV transport of molecular cargo to resident and distant cells. Most studies on EV-mediated signaling have been performed in two-dimensional (2D) monolayer cell cultures, largely because of their simplicity and high-throughput screening capacity. Three-dimensional (3D) cell cultures can be used to study cell-to-cell and cell-to-matrix interactions, enabling the study of EV-mediated cellular communication. 3D cultures may best model the role of EVs in formation of the tumor microenvironment (TME) and cancer cell-stromal interactions that sustain tumor growth. In this review, we discuss EV biology in 3D culture correlates of the TME. This includes EV communication between cell types of the TME, differences in EV biogenesis and signaling associated with differing scaffold choices and in scaffold-free 3D cultures and cultivation of the premetastatic niche. An understanding of EV biogenesis and signaling within a 3D TME will improve culture correlates of oncogenesis, enable molecular control of the TME and aid development of drug delivery tools based on EV-mediated signaling.

Inorganic phosphate (Pi) has been recognized as an important signaling molecule that modulates chondrocyte maturation and cartilage mineralization. However, conclusive experimental evidence for its involvement in early chondrogenesis is still lacking. Here, using high-density monolayer (2D) and pellet (3D) ATDC5 chondrogenic models treated with ITS+, β-glycerophosphate (βGP), or ITS+/βGP, we demonstrate that the cell response to Pi does not correlate with the Pi concentration in the culture medium but is better predicted by the availability of Pi on a per cell basis (Pi abundance). Three levels of abundance were found: basal (Pi/DNA < 10 ng/μg), moderate (Pi/DNA=25.3 – 32.3 ng/μg), and high abundance (Pi/DNA > 60 ng/μg). In chondrogenic medium alone, the abundance levels were at the basal level in 2D culture and moderate in 3D cultures. The addition of 10mM βGP resulted in moderate abundance in 2D and high in 3D cultures. Moderate Pi abundance enhanced early chondrogenesis and production of aggrecan and type II collagen whereas high Pi abundance inhibited chondrogenic differentiation and induced rapid mineralization. Inhibition of sodium phosphate transporters also reduced expression of chondrogenic markers. When 3D ITS+/βGP cultures were treated with levamisole to reduce ALP activity, Pi abundance was decreased to moderate levels, which resulted in significant upregulation of chondrogenic markers, similar to the response in 2D cultures. Delay of phosphate delivery until after early chondrogenesis occurs (7 days) no longer enhanced chondrogenesis, but instead accelerated hypertrophy and mineralization. Together, our data highlights the dependence of chondroprogenitor cell response to Pi on its availability to individual cells and the chondrogenic maturation stage of these cells and suggest that appropriate temporal delivery of phosphate to ATDC5 cells in 3D cultures represents a rapid model for mechanistic studies into the effects of exogenous cues on chondrogenic differentiation, chondrocyte maturation, and matrix mineralization.

Repeating L- and D-chiral configurations determine polylactide (PLA) stereochemistry which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries cause differential immune responses in-vivo, this observation has yet to be effectively modeled in-vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing > 99% L-lactide (PLLA), > 99% D-lactide (PDLA) and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1b, TNF-a and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Whereas both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries, and underscore the pivotal role of immunometabolism on the biocompatibility of biomaterials applied in medicine.

Repeating l- and d-chiral configurations determine polylactide (PLA) stereochemistry, which affects its thermal and physicochemical properties, including degradation profiles. Clinically, degradation of implanted PLA biomaterials promotes prolonged inflammation and excessive fibrosis, but the role of PLA stereochemistry is unclear. Additionally, although PLA of varied stereochemistries causes differential immune responses in vivo, this observation has yet to be effectively modeled in vitro. A bioenergetic model was applied to study immune cellular responses to PLA containing >99% l-lactide (PLLA), >99% d-lactide (PDLA), and a 50/50 melt-blend of PLLA and PDLA (stereocomplex PLA). Stereocomplex PLA breakdown products increased IL-1β, TNF-α, and IL-6 protein levels but not MCP-1. Expression of these proinflammatory cytokines is mechanistically driven by increases in glycolysis in primary macrophages. In contrast, PLLA and PDLA degradation products selectively increase MCP-1 protein expression. Although both oxidative phosphorylation and glycolysis are increased with PDLA, only oxidative phosphorylation is increased with PLLA. For each biomaterial, glycolytic inhibition reduces proinflammatory cytokines and markedly increases anti-inflammatory (IL-10) protein levels; differential metabolic changes in fibroblasts were observed. These findings provide mechanistic explanations for the diverse immune responses to PLA of different stereochemistries and underscore the pivotal role of immunometabolism in the biocompatibility of biomaterials applied in medicine.