Biomaterial-Based Metabolic Regulation in Regenerative Engineering

Abstract

Recent advances in cell metabolism studies have deepened the appreciation of the role of metabolic regulation in influencing cell behavior during differentiation, angiogenesis, and immune response in the regenerative engineering scenarios. However, the understanding of whether the intracellular metabolic pathways could be influenced by material-derived cues remains limited, although it is now well appreciated that material cues modulate cell functions. Here, an overview of how the regulation of different aspect of cell metabolism, including energy homeostasis, oxygen homeostasis, and redox homeostasis could contribute to modulation of cell function is provided. Furthermore, recent evidence demonstrating how material cues, including the release of inherent metabolic factors (e.g., ions, regulatory metabolites, and oxygen), tuning of the biochemical cues (e.g., inherent antioxidant properties, cell adhesivity, and chemical composition of nanomaterials), and changing in biophysical cues (topography and surface stiffness), may impact cell metabolism toward modulated cell behavior are discussed. Based on the resurgence of interest in cell metabolism and metabolic regulation, further development of biomaterials enabling metabolic regulation toward dictating cell function is poised to have substantial implications for regenerative engineering.

Keywords: biomaterials; energy metabolism; metabolic regulation; metabonegenic regulation; regenerative engineering.

Figure 1

Overview of the core cell metabolic pathways. The energy source, ATP, is imperative for cell survival, proliferation, differentiation, and cell‐specific functions. ATP generation is derived from the intracellular processes, glycolysis, and cellular respiration. Glucose, a primary energy substrate, is imported into the cytosol, from the extracellular space and undergoes conversion into pyruvate via a series of chemical reactions collectively known as glycolysis obtaining a net of 2 ATP per mole of glucose. Pyruvate is converted into acetyl–CoA (Ac–CoA) in mitochondrial to enter the tricarboxylic acid (TCA) cycle, which drives the electron transfer chain (ETC) yielding a net production of 36 ATP molecules in a process called oxidative phosphorylation (OXPHOS). Notably, in certain cell types, such as cancer and endothelial cells, glycolysis‐derived pyruvate molecules are converted into lactate even when ample O₂ is available, a phenomenon referred to as aerobic glycolysis. The glycolytic flux can also be directed through the pentose phosphate pathway (PPP) to generate NADPH, a cofactor for redox homeostasis and cellular respiration, as well as ribose‐5‐phosphate, a substrate for nucleotide synthesis. Furthermore, alternative macromolecules can feed into the TCA cycle besides pyruvate exhibited by conversion of fatty acids to acetyl–CoA via β‐oxidation and by conversion of glutamine to α‐ketoglutarate (α‐KG) via glutaminolysis. TCA cycle citrate may also be exported to the cytosol where it serves as a substrate for itaconic acid synthesis in M1 macrophages or may be converted to acetyl–CoA for fatty acid synthesis. Nucleocytoplasmic acetyl–CoA is additionally required as a substrate for histone acetylation (Ac) of chromatin histones, which impacts chromatin structure and gene transcription.

Figure 2

Oxygen‐dependent regulation of hypoxia‐induced factor (HIF) signaling. A) In normoxic environment, prolyl hydroxylase domain enzymes (PHDs) hydroxylate HIF‐1α subunits with production of carbon dioxide (CO₂) and succinate as byproducts. Hydroxylation requires α‐KG and oxygen (O₂) as substrates and is catalyzed by ferrous ions (Fe²⁺). The hydroxylated HIF‐1α subunits then undergo ubiquitination and degradation preventing nuclear translocation. B) In hypoxic conditions, there is an insufficient O₂ supply for extensive hydroxylation of HIF‐1α subunits avoiding degradation to translocate to the nucleus and complex with HIF‐1β to promote transcription.

Figure 3

Regulation of redox homeostasis. Primary intracellular production of radical oxygen species (ROS) is derived from the metabolic mitochondrial ETC and membrane‐bound NADPH oxidase (NOX). Enzymes, such as superoxide dimutases (SODs) and catalase, in conjunction with antioxidant molecules, reduced glutathione (GSH), and NADPH, perform critical roles in the endogenous antioxidant defense system to preserve redox homeostasis. Anion superoxide (O₂•⁻) is the leading form of produced ROS, which is rapidly converted into cell permeable hydrogen peroxide (H₂O₂) by SOD2 in the mitochondria, by SOD1 in the cytosol, and extracellularly by SOD3. H₂O₂ can be catalyzed to the most reactive hydroxyl radicals (HO•) in the presence of Fe²⁺ (Fenton reaction) or be converted into water (H₂O) by catalase. The reduced form of glutathione (GSH) and the oxidized form of glutathione (GSSG), together with a reducing agent (e.g., NADPH) represent another major antioxidant mechanism converting radical H₂O₂ to H₂O.

Figure 4

Overview of biomaterial‐based regulation of cell metabolism. Metabolic regulation from biomaterials design may be achieved by A) releasing metabolic regulators (e.g., metal ions, metabolites, and oxygen), which subsequently enter cells to modulate intracellular metabolic activities; by B) introducing antioxidative properties (e.g., ion chelation or ROS “quenching”) to or altering cell adhesivity of the materials through chemical modification to modulate external factors indirectly impacting intracellular redox homeostasis; and by C) modulating the biophysical properties of the base materials via the design of surface features and stiffness alteration to provide biophysical cues which are converted to biochemical cues involved in metabolic regulation.

Figure 5

Metabolic regulator release from biomaterials. A) i) Schematic illustration of the citrate‐mediated metabonegenic mechanism in a human mesenchymal stem cell (hMSC) resulting from citrate release upon citrate‐based biomaterial degradation, ii) elevated intracellular ATP levels via modulation of the major energy‐producing pathways (e.g., glycolysis and OXPHOS) by citrate, which subsequently iii) promoted the Runx2 mediated osteo‐differentiation.80 B) Inorganic phosphate (Pi) released from resorbable calcium phosphate was found to i) enter cells to reach the mitochondria where it serves as the direct substrate for ATP syntheses. The cumulated ATP is secreted from cells and ii) degraded to adenosine, which in turn impacts osteogenesis as exhibited by iii) osteocalcin production via autocrine/paracrine signaling (scale bar: 100 µm) (Adapted with permission.19 Copyright 2013, PNAS). C) Oxygen‐generating materials can be designed by embedding oxygen‐forming compounds, like CaO₂, into hydrophobic polymers, such as PDMS. A PDMS disk containing 25% w/w CaO₂ after soaking in buffer saline could lead to sufficient oxygen generation up to 6 weeks (Adapted with permission.100 Copyright 2012, PNAS).

Figure 6

Biochemical cues from biomaterials for metabolic regulation. A) Citrate‐based biodegradable elastomers with inherent antioxidant properties poly(octamethylene citrate ascorbate) (POCA) (upper) was developed to possess (middle) strong radical scavenging activity and (lower) potent Fe²⁺ chelating capability (Adapted with permission.93 Copyright 2014, Elsevier). B) Cerium oxide nanoparticle (CONP)‐alginate composite hydrogel was developed for the encapsulation of β cells in which system CONP provided ubiquitous and renewable antioxidant protection from external ROS damage, resulting in greatly improved survival of β cells under superoxide exposure (scale bars: 200 µm) (Adapted with permission.109 Copyright 2011, RSC Publishing). C) Ultrathin polyelectrolyte multilayer ([PDADMA/PSS, 1.0]10) was designed to coat tissue culture plastic serving as a “biocompatible” but poorly adhesive substrate on which 3T3 fibroblasts exhibited a rounded morphology, diffuse organization of the actin cytoskeleton, stunted proliferation together with heightened metabolic stress (left) compared to that on tissue culture plastic alone (right) (scale bars: 10 µm). (Adapted with permission.102 Copyright 2018, ACS). D) Schematic illustration of how uptake of graphene nanosheets impairs the migration and invasion of metastatic breast cancer cells by disturbing electron transfer in the ETC and thereby reducing ATP production (Adapted with permission.103 Copyright 2014, Elsevier).

Figure 7

Biophysical cues from biomaterials for metabolic regulation. A) MG63 cells cultured on micropillars topography with a dimension of 5 µm × 5 µm × 5 µm and a spacing of 5 µm was found to drastically alter actin cytoskeleton organization and to induce attempted caveolae‐mediated phagocytosis of beneath micropillar evidenced by elevated caveolin‐1 expression and activation, which was accompanied with increased ROS and reduced ATP production leading to suppressed osteogenesis, as compared to that on flat surface (Adapted with permission.115 Copyright 2016, Elsevier). B) Supramolecular hydrogels of simple chemical functionality with tunable stiffness were designed to reveal stiffness‐related differentiation of pericytes/MSCs toward different lineages. In combination with metabolomics analysis, two types of lipid, the lysophosphatidic acid (GP18:0) in the glycerolipid pathway and the cholesterol sulfate (CS) in the steroid biosynthesis pathway, were identified and validated as key regulatory metabolites that may be involved in direct chondrogenic (shown as SOX‐9 expression) and osteogenic differentiation (shown as osteopontin expression), respectively (Adapted with permission.116 Copyright 2016, Elsevier).