Microenvironment-Responsive Nanomaterials for Colorectal Anastomotic Healing: A Signal–Threshold–Response Framework for Stage-Adaptive Repair

Abstract

Colorectal anastomotic leakage remains a major source of morbidity and mortality after intestinal reconstruction. Many current sealants, hydrogels, nanocomposites, and local nanomedicine platforms are optimized for static mechanical support, tissue adhesion, or sustained release, whereas the anastomotic wound evolves through temporally coupled changes in perfusion, redox state, protease activity, pH, and microbial activity. This review evaluates stimuli-responsive nanomaterials through an anastomosis-specific signal–threshold–response framework. Evidence is separated into direct preclinical studies performed in colorectal or colonic anastomosis models and transferable mechanistic or proof-of-concept evidence derived from gastrointestinal and other non-anastomotic tissue-repair models. We first synthesize available evidence on the spatial distribution, postoperative dynamics, and pathophysiological roles of anastomosis-relevant cues. We then evaluate pH-, reactive oxygen species-, enzyme-, hypoxia-, microbial-, and multi-input-responsive systems according to their sensing mechanisms, reported activation conditions, material transformations, therapeutic outputs, and levels of evidence. We further propose design principles for matching response kinetics and mechanical evolution to the inflammatory, proliferative, and remodeling phases. Future development should prioritize quantitative mapping of local cues, logic-gated or sequential activation, wet-tissue mechanics, microbiota-preserving antimicrobial action, and validation in clinically relevant large-animal models. This framework positions responsive nanomaterials as stage-adaptive adjuncts rather than generic drug-delivery systems for colorectal anastomotic repair.

Introduction

Clinical Burden of Anastomotic Leakage (AL)

Intestinal reconstruction is routinely performed for colorectal cancer and inflammatory bowel disease, and successful anastomotic healing is essential for postoperative recovery. Despite advances in surgical techniques and perioperative care, anastomotic leakage (AL) still occurs in 5–10% of patients and is associated with increased perioperative mortality, prolonged hospitalization, reoperation, and a higher risk of long-term tumor recurrence.^1–4 Its clinical impact is particularly pronounced after low rectal anastomosis, where leakage can compromise sphincter-preserving treatment and postoperative quality of life.^5,6 Current preventive strategies mainly aim to improve mechanical integrity through optimized anastomotic techniques, reinforcement sutures, external patches, and biological sealants.^7–10 Although these approaches may reduce structural failure, they do not directly correct the biological disturbances that impair tissue repair. Clinical and experimental evidence increasingly indicates that AL represents a local failure of healing shaped by both anastomotic microenvironmental abnormalities and systemic host factors, rather than mechanical dehiscence alone.^11–13 Therefore, effective nanomaterial strategies should combine mechanical support with active regulation of the local healing environment.

Anastomotic Healing as a Dynamic Microenvironmental Process

Colorectal anastomotic healing proceeds through overlapping inflammatory, proliferative, and remodeling phases, each characterized by a distinct but evolving set of biological signals (Figure 1).^14,15 Successful repair depends not only on the presence of cytokines, growth factors, extracellular matrix (ECM) components, and adequate perfusion, but also on their timing and magnitude.¹⁶ These temporal changes provide potential triggers for responsive nanomaterials; however, the same signals may support physiological repair at moderate levels and become harmful when excessive or prolonged.

Figure 1.

Overlapping phases and qualitative temporal dynamics of colorectal anastomotic healing. Colorectal anastomotic repair proceeds through overlapping inflammatory, proliferative, and remodeling phases rather than strictly sequential stages. Early healing is characterized predominantly by tissue hypoxia, neutrophil recruitment, reactive oxygen species (ROS) generation, and inflammatory signaling. The proliferative phase is associated with fibroblast migration and proliferation, extracellular matrix (ECM) deposition, and angiogenesis, whereas later remodeling involves collagen maturation, matrix reorganization, and regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). The lower schematic summarizes the qualitative temporal predominance of hypoxia, ROS, protease activity, angiogenesis, collagen deposition, and matrix remodeling across healing. These profiles indicate relative temporal trends and should not be interpreted as quantitative concentration–time curves. ↑ and ↓, where used, indicate relative increases and decreases, respectively.

During the early postoperative phase, surgical trauma and disruption of local blood supply induce a transient ischemic and hypoxic microenvironment at the anastomotic site. This rapidly triggers the innate immune response.^17–19 Transient ROS production contributes to antimicrobial defense and cell signaling, whereas sustained oxidative stress damages cellular macromolecules, amplifies inflammation, and impairs tissue repair.^18–20 Accordingly, indiscriminate or premature ROS elimination may weaken host defense, while delayed intervention may fail to prevent oxidative injury. Intestinal manipulation and bowel preparation can simultaneously disturb microbial homeostasis, favoring the expansion of opportunistic pathogens.^21,22 At the wound surface, biofilm formation and the release of lipopolysaccharides, collagenases, and other virulence factors can prolong inflammation and degrade newly deposited matrix.²³ Collagenase-producing bacteria, including Enterococcus faecalis, are particularly relevant because they can directly reduce anastomotic mechanical strength.^22,23 Increased pathogenic bacterial burden and biofilm formation have also been reported in patients who develop AL.^21,24 Thus, hypoxia, excessive ROS, bacterial enzymes, and pathogen-associated signals represent early-stage material targets, but therapeutic responses must preserve the physiological functions of inflammation and commensal microbiota.

During the proliferative phase, fibroblast migration, myofibroblast differentiation, ECM deposition, and angiogenesis become dominant. These processes are regulated by vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), transforming growth factor-β (TGF-β), and other repair-associated signals.^25,26 Newly formed micro vessels improve oxygen and nutrient delivery, while collagen and fibronectin deposition gradually restore anastomotic strength.²⁷ Persistent hypoxia or inflammation at this stage delays the transition from tissue defense to regeneration, whereas premature suppression of growth-factor signaling or angiogenesis can impair granulation tissue formation and increase leakage risk.²⁵ Biomaterials intended for this phase should therefore shift from early antimicrobial and anti-inflammatory activity toward controlled support of vascularization, fibroblast function, and matrix synthesis.

Remodeling is governed largely by matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), which coordinate collagen degradation, replacement, and fiber reorganization.^28,29 Early type III collagen is progressively replaced by mechanically stronger type I collagen, and fibers align along local tension axes.^15,28 Excessive MMP activity accelerates collagen loss and weakens the anastomosis, whereas insufficient activity may cause excessive matrix accumulation, fibrosis, and reduced tissue compliance.³⁰ MMPs should therefore be regarded as regulatory signals rather than uniformly harmful targets. Effective enzyme-responsive systems must modulate abnormal proteolysis without blocking the matrix turnover required for scar maturation. More broadly, responsive nanomaterials should be matched to the sequence of postoperative signals: early hypoxia, oxidative stress, and microbial activity; subsequent angiogenic and matrix-forming signals; and later MMP-dependent remodeling. Quantitative human data on the intensity and duration of these signals remain limited, which is a major constraint on defining clinically relevant activation thresholds.

Rationale for Stimuli-Responsive Nanomaterials

Conventional nanomaterials for anastomotic repair generally provide predetermined mechanical support, adhesion, or drug release and cannot adapt as the wound environment changes.³¹ In contrast, postoperative anastomoses undergo temporal changes in pH, oxygenation, ROS, protease activity, microbial burden, and tissue mechanics.³² A mismatch between these signals and material behavior may cause premature degradation, nonspecific cargo release, inadequate reinforcement, or interference with physiological repair.

Stimuli-responsive nanomaterials contain chemical bonds or structural domains that change in response to local cues.³³ Acid-labile linkages can trigger cargo release at reduced pH, ROS-sensitive groups can regulate degradation or drug release, and MMP-cleavable peptides can alter material behavior when protease activity increases.^34,35 Their benefit depends on whether the activation range and response kinetics match the magnitude and duration of the target signal. Because physiological ROS and MMP activity also support antimicrobial defense and matrix turnover, responsive systems should modulate excessive or persistent signals rather than suppress them indiscriminately. Previous reviews have discussed hydrogel adhesives, external reinforcement, multifunctional patches, and infection-directed strategies,^36–39 but have mainly organized evidence by material type or therapeutic function. This review instead adopts an anastomosis-specific signal–threshold–response framework and distinguishes direct preclinical colorectal/colonic anastomosis evidence from transferable mechanistic or proof-of-concept evidence derived from other experimental models.

Literature Search and Evidence Selection

This review was designed as a mechanism-oriented narrative review rather than a systematic review or meta-analysis. To improve the transparency and reproducibility of evidence identification, a structured literature search was conducted using PubMed/MEDLINE and Web of Science Core Collection, covering database inception through April 30, 2026. Two complementary search strategies were used to reflect the evidence structure of this review. The first targeted studies directly related to colorectal, colonic, or intestinal anastomotic healing and combined terms including “colorectal anastomosis”, “colonic anastomosis”, “intestinal anastomosis”, “anastomotic healing”, and “anastomotic leakage” with material-related terms such as “nanomaterial”, “nanoparticle”, “hydrogel”, “biomaterial”, “adhesive”, “sealant”, and “stimuli-responsive”. The second strategy was used to identify mechanistically relevant responsive-material studies by combining terms related to pH, ROS, proteases or MMPs, hypoxia, microbial activity, and enzymes with terms including “responsive material”, “nanomaterial”, “nanoparticle”, “hydrogel”, “drug delivery”, “wound healing”, and “tissue repair”.

Titles and abstracts were screened for relevance, followed by assessment of the full text of potentially eligible articles. Reference lists of relevant reviews and included studies were additionally examined to identify studies not retrieved by the database searches. Original studies were prioritized when extracting material composition, activation conditions, response or release kinetics, mechanical properties, and biological outcomes. Review articles were used primarily for background information and citation tracing rather than as the sole source of quantitative material parameters.

Evidence was selected according to its proximity to the intended application. Studies directly evaluating material platforms in colorectal or colonic anastomotic models were considered the most application-specific evidence and are summarized in Table 1. Gastrointestinal or non-anastomotic studies were included when they provided experimentally demonstrated responsive mechanisms, activationconditions, or material behaviors relevant to anastomosis-specific design; these studies are presented separately in Supplementary Table S1 and are not interpreted as direct evidence of efficacy in colorectal anastomotic healing. Studies were excluded when they did not provide information relevant to anastomotic biology, responsive-material mechanisms, or transferable material design. Purely oncological or other disease-specific applications were considered only when they provided mechanistic information on the responsive chemistry itself and were not used as evidence of colorectal anastomotic efficacy.

Table 1.

Representative Responsive and Microenvironment-Modulating Material Platforms Directly Evaluated in Colorectal or Colonic Anastomotic Repair

Study	Exact Material/Platform	Design Principle and Trigger or Mode of Action	Reported Activation Threshold/Range	Response/Release Kinetics	Therapeutic Payload	Experimental Model	Principal Mechanical and Biological/Healing Outcomes	Evidence Level and Critical Appraisal	References
Reischl et al, 2021	Collagen-IV-targeted polymeric nanoparticles loaded with the Annexin A1 biomimetic peptide Ac2-26	Collagen-IV targeting promotes localization to injured tissue; Ac2-26 promotes resolution of inflammation through suppression of pro-inflammatory/NF-κB signaling. This is a targeted pro-resolving system rather than an endogenous cue-triggered platform.	N/A	NR	Ac2-26	Murine preoperative DSS-colitis model followed by intestinal anastomosis	Mechanical outcome: NR. Biological/healing outcomes: improved preoperative colitis activity, postoperative histological healing scores, and weight recovery; associated with reduced pro-inflammatory signaling and changes in macrophage phenotype.	Direct in vivo murine anastomosis evidence. Strong disease-relevant inflammatory model, but no endogenous cue-dependent activation or quantitative tissue threshold was demonstrated.	[40]
Lee et al, 2024	P-C-Col IV-Ac2-26-NPs: Ac2-26-loaded PLGA nanoparticles with short PEGylated/collagen-IV-targeted surface, sequentially coated with chitosan and pectin	Pectin/chitosan protect the nanoparticle during upper-GI transit and are degraded by colonic microbiota, releasing collagen-IV-targeted Ac2-26 nanoparticles near the colonic wound. Primarily regional microbiota-mediated targeting, rather than pathology-specific activation.	Quantitative microbial enzyme/activity threshold: NR	In PBS at 37 °C, pectin/chitosan-coated NPs released approximately 20% peptide at 24 h, ~38% at 72 h, and ~60% over 7 days; these values describe formulation release rather than microbiota-triggered kinetics	Ac2-26	Murine model with 7-day 2% DSS-induced colitis followed by microsurgical colonic anastomosis; oral treatment every 3.5 days	Mechanical outcome: NR. Biological/healing outcomes: reduced postoperative colitis activity; improved macroscopic wound closure, intra-abdominal adhesion scores, and microscopic healing scores.	Direct in vivo murine colonic-anastomosis evidence. One of the clearest direct nanomedicine examples. However, colonic microbial degradation is not specific to pathological leakage, and no quantitative microbial activation threshold was established.	[41]
Alric et al, 2024	20% Pluronic F127 thermoresponsive hydrogel containing adipose-derived stromal-cell extracellular vesicles (ADSC-EVs; final concentration 1 × 1011 EVs/mL; mean EV size ~126 nm)	Temperature-induced sol-to-gel transition provides local EV retention; progressive dilution allows subsequent EV release	Liquid at <20 °C and gel transition at >20 °C; dilution below approximately 13% PF-127 induces gel-to-solution transition	Gradual EV delivery through progressive gel dilution; exact in vivo release rate NR	ADSC-derived EVs	40 male SD rats; colorectal irradiation (3 × 12.5 Gy) followed 3 weeks later by low colo-colonic anastomosis	Mechanical outcome: NR. Biological/healing outcomes: EV + gel treatment produced smaller anastomotic ulcers, less fibrosis, and reduced inflammatory infiltrates.	Direct in vivo high-risk rat colonic-anastomosis evidence. Quantitative physical transition conditions were reported. The trigger is temperature/dilution rather than a pathological anastomotic cue.	[42]
Wang et al, 2025	In situ hydrogel formed from oxidized hyaluronic acid (OHA), adipic-acid-dihydrazide-modified hyaluronic acid (HA-ADH), ε-poly-L-lysine (ε-PLL), and tannic acid (TA)	Wet-tissue adhesion and physical sealing combined with intrinsic antibacterial and ROS-scavenging activities; microenvironment modulation is constitutive rather than ROS-triggered	N/A	Described as rapid gelation; exact gelation time NR in the accessible primary report	None as a separately loaded drug; ε-PLL and TA provide intrinsic bioactivity	Colon-resection anastomotic-leak models in rats and rabbits; rat cecum–abdominal-wall adhesion model	Mechanical/functional outcomes: wet-tissue adhesion and effective sealing of anastomotic leakage. Biological/healing outcomes: reduced postoperative adhesion, antibacterial and antioxidant effects, and promotion of anastomotic healing.	Direct multi-species preclinical evidence. Strong direct relevance to colorectal AL, but should be classified as redox/microenvironment-modulating rather than ROS-responsive because endogenous ROS does not control material activation.	[43]
Ju et al, 2025	Temperature-sensitive extracellular-matrix hydrogel based on porcine small-intestinal-submucosa (SIS) ECM physically modified with boric acid and 4-ARM-PEG-SC	Injectable hydrogel provides mechanical sealing, digestive-fluid resistance, antibacterial, antioxidant, and anti-inflammatory activity, while supporting vascularization and tissue repair	Exact sol–gel transition temperature/range: NR	NR	No separately loaded therapeutic payload reported	Rat colonic anastomotic-leak model	Mechanical/functional outcomes: tissue adhesion, sealing, and resistance to digestive-fluid exposure. Biological/healing outcomes: improved the local anastomotic microenvironment, promoted cell proliferation and vascular formation, reduced pro-inflammatory factors, and reduced colonic AL incidence.	Direct in vivo rat evidence. Multifunctional and directly relevant, but the reported temperature responsiveness is primarily a deployment/gelation property rather than pathology-selective sensing.	[44]
Zhu et al, 2025	PGOT hydrogel composed of γ-poly(glutamic acid) conjugated with L-cysteine and dopamine (γ-PGA-Cys-DA), oxidized konjac glucomannan (OKGM), and tannic acid	Dynamic thiol–aldehyde crosslinking enables rapid gelation/self-healing; catechol/polyphenol groups provide wet adhesion; intrinsic antibacterial, pro-angiogenic, and immunomodulatory effects	N/A for endogenous pathological cue	Gelation approximately 12s	None as a separately loaded drug	Rat colorectal anastomosis	Mechanical/functional outcomes: rapid self-healing gel formation and wet-tissue adhesion; gelation occurred in approximately 12s. Biological/healing outcomes: reduced AL incidence and accelerated regenerative healing, with antibacterial activity, enhanced angiogenesis, and an increased reparative macrophage-associated phenotype.	Direct in vivo rat colorectal-anastomosis evidence. Provides rapid mechanical sealing plus biological modulation, but is not activated by a quantified local pathological signal.	[45]
Cira et al, 2025	Chitosan/pectin-coated, collagen-IV-targeted Ac2-26 nanoparticles, (pc)-Col-IV-Ac2-26-NPs	Collagen-IV wound targeting plus pro-resolving Ac2-26 delivery; oral coating provides microbiota-dependent regional release; systemic, oral, and rectal delivery routes were compared	Quantitative cue threshold: NR	NR in the accessible primary report	Ac2-26	Acute Crohn’s-disease-like colitis in mice with distal end-to-end colo-colonic anastomosis; intraperitoneal, oral, and rectal delivery evaluated	Mechanical outcome: NR. Biological/healing outcome: enhanced anastomotic healing in inflamed tissue and demonstrated feasibility of route-dependent targeted perioperative nanotherapy.	Direct in vivo murine high-risk anastomosis evidence. Extends the Ac2-26 platform to Crohn-like inflammation and compares delivery routes, but does not establish an endogenous cue–threshold relationship.	[46]
Antonelli et al, 2025	Acellular urinary-bladder-matrix (UBM) ECM hydrogel applied directly to the rectal anastomosis	Physical coverage combined with ECM-mediated constructive tissue remodeling; non-responsive microenvironment-modulating biomaterial	N/A	N/A/NR	None	Female SD rats; standardized distal colonic resection and sutured low rectal anastomosis below the peritoneal reflection	Mechanical outcome: NR. Biological/healing outcomes: leak rate decreased from 46% in controls to 8% with UBM hydrogel, with fewer perianastomotic adhesions and an improved composite histologic healing score.	Direct in vivo rat low-rectal-anastomosis evidence. Strong direct healing data and useful comparator, but this is an ECM biomaterial rather than an intrinsically responsive or nanoscale platform.	[47]
Deng et al, 2026	Cold-atmospheric-plasma-activated hydrogel (Hyd@CAP); exact base-polymer composition should be inserted after full-text verification	Local delivery and sustained release of exogenous reactive oxygen and nitrogen species (RONS); antimicrobial, anti-inflammatory, and tissue-reparative effects	N/A: endogenous ROS/RONS do not trigger activation	Sustained RONS release reported; quantitative release rate/half-life NR in the accessible primary report	CAP-generated RONS	SD rat colonic anastomotic-leak model	Mechanical/functional outcomes: stable coverage and adhesion at the anastomosis. Biological/healing outcomes: reduced postoperative adhesion scores, antimicrobial and anti-inflammatory effects, and accelerated intestinal epithelial regeneration associated with mTOR activation.	Direct in vivo rat CAL evidence. Should be classified as redox-modulating, not ROS-responsive. Therapeutic window and local RONS dose–toxicity relationship requires quantitative definition.	[48]
Yu et al, 2026	TNGD thermosensitive amphoteric nanogels synthesized from N-isopropylacrylamide (NIPAM) and methacryloylated arginine (M-Arg), followed by dopamine conjugation; nanogels aggregate into a thermosensitive aggregated hydrogel (TAH)	Heating above the volume-phase-transition temperature (VPTT) induces nanogel phase transition and supramolecular aggregation through hydrophobic, hydrogen-bonding, and electrostatic interactions; intrinsic anti-inflammatory, antibacterial, and antifouling functions	Above formulation-specific VPTT; exact numerical VPTT NR here and should be extracted from the full article before final typesetting	Aggregation increases with temperature/heating duration; exact transition time NR	None	Diabetic rat colonic-anastomosis model	Mechanical/anastomotic-strength outcome: NR. Biological/functional outcomes: improved the diabetic inflammatory microenvironment and reduced the incidence/severity of postoperative tissue adhesions compared with commercial anti-adhesion materials.	Direct in vivo diabetic colonic-anastomosis evidence and a genuine nanogel-based platform. Primary outcome was postoperative adhesion rather than AL or anastomotic tensile healing, so relevance to leakage prevention remains indirect.	[49]

Notes: “Activation threshold/range” refers to the stimulus level experimentally required to alter the material itself. It should not be interpreted as the physiological or pathological threshold of the corresponding cue in human colorectal anastomotic tissue unless such a value was measured directly at the anastomotic tissue–material interface. Temperature-induced gelation, constitutive ROS scavenging, and delivery of exogenous RONS are therefore distinguished from endogenous pathology-responsive activation. No human clinical efficacy study was identified among the directly evaluated platforms summarized here. Mechanical outcomes were reported using the study-specific measures available in the original publications. NR indicates that a directly comparable mechanical endpoint, such as wet-tissue adhesion strength, bursting pressure, tensile strength, or cyclic mechanical stability, was not quantitatively reported. Mechanical data obtained using different test configurations were not normalized or directly ranked across studies. Evidence appraisal considered model relevance, demonstration of cue-dependent activation, quantitative characterization of activation/response, comparator design, and relevance of the reported healing outcomes. The descriptors are intended as a qualitative appraisal of preclinical evidence rather than a formal clinical evidence grade.

Abbreviations: NR, not reported quantitatively in the primary source used for extraction; N/A, not applicable because activation does not depend on an endogenous pathological cue; AL, anastomotic leakage; CAP, cold atmospheric plasma; DSS, dextran sodium sulfate; ECM, extracellular matrix; EV, extracellular vesicle; HA, hyaluronic acid; RONS, reactive oxygen and nitrogen species; UBM, urinary bladder matrix.

To assess the strength and applicability of the evidence, studies were evaluated qualitatively according to predefined domains rather than assigned a pooled numerical quality score. These domains included: (i) proximity of the experimental model to colorectal anastomotic repair; (ii) whether cue-dependent material activation was directly demonstrated; (iii) whether activation conditions and response kinetics were quantitatively characterized; (iv) the presence of appropriate material or treatment comparators; and (v) the relevance of the reported outcomes to anastomotic healing, including leakage, mechanical integrity, tissue repair, inflammation, and microbial effects. Evidence was therefore interpreted hierarchically, with direct in vivo colorectal or colonic anastomosis studies given the greatest application-specific weight, followed by gastrointestinal but non-anastomotic studies, non-gastrointestinal tissue-repair models, and mechanistic or disease-specific studies used primarily to support responsive chemistry. This hierarchy reflects model relevance and mechanistic validation and should not be interpreted as a formal clinical evidence-grading system. Throughout this review, the term “direct anastomotic evidence” is reserved for studies performed in colorectal or colonic anastomosis models, whereas findings from gastrointestinal non-anastomotic, wound-healing, tumor, or other tissue-repair models are described as mechanistic proof-of-concept or transferable preclinical evidence and are discussed only in terms of their potential relevance to colorectal anastomotic repair.

Microenvironmental Cues Relevant to Nanomaterial Design

Colorectal anastomotic healing is shaped by spatially and temporally varying changes in perfusion, metabolism, redox balance, protease activity, and microbial exposure (Figure 2). These cues may support normal repair when transient and appropriately regulated but impair healing when excessive or persistent. This section examines five cues relevant to biomaterial design: hypoxia and metabolic stress, local pH changes, ROS, protease activity, and microbe-derived molecular signals.

Figure 2.

Engineering-relevant microenvironmental cues in colorectal anastomotic healing: physiological adaptation versus pathological persistence. Hypoxia, local pH changes, ROS, protease activity, and microbe-derived signals vary spatially and temporally during colorectal anastomotic repair. Their biological effects depend on signal magnitude and duration. Transient or moderate changes may contribute to physiological adaptation, host defense, extracellular-matrix turnover, and tissue repair, whereas excessive or persistent signals may promote oxidative injury, sustained inflammation, collagen degradation, matrix instability, or impaired healing. The schematic therefore distinguishes physiological/adaptive states from pathological/maladaptive states for each cue. Local acidification is shown conceptually and does not imply that luminal, drainage-fluid, and tissue-interstitial pH values are interchangeable. Similarly, microbial signals reflect both background commensal exposure and potentially harmful microbial activity at the wound interface. The indicated engineering strategies represent candidate material-design targets and do not by themselves establish cue-dependent activation in colorectal anastomotic tissue. ↑ and ↓ indicate relative increases and decreases, respectively; directional arrows indicate biological or engineering relationships rather than quantitative effect sizes.

Hypoxia and Metabolic Stress

Surgical transection and disruption of local blood vessels reduce perfusion and oxygen tension at the anastomotic site, thereby activating hypoxia-inducible factor (HIF) signaling.⁵⁰ In the LekCheck study, anemia was associated with an increased risk of colorectal anastomotic leakage, supporting the clinical importance of adequate oxygen delivery; however, this association does not define local oxygen tension at the anastomosis or the threshold required to activate a hypoxia-responsive material.⁵¹

HIF activity is regulated by prolyl hydroxylases (PHDs), which promote HIF-α degradation under normoxic conditions. In murine ischemia and sepsis models, PHD2 haploinsufficiency or pharmacological PHD inhibition stabilized HIF-1α, promoted a pro-reparative macrophage phenotype, increased collagen deposition and bursting pressure, and reduced AL incidence.⁵⁰ HIF-1α also induces vascular endothelial growth factor, glucose transporter 1 (VEGF1), and glycolytic enzymes, shifting cellular metabolism toward glycolysis.^52–54 The resulting lactate accumulation can influence angiogenesis and macrophage function.^55,56 These responses may support early adaptation to reduced oxygen availability, whereas persistent hypoxia impairs the prolyl and lysyl hydroxylation required for collagen maturation, suppresses fibroblast activity, and reduces ECM deposition.⁵⁷

For nanomaterial design, hypoxia can serve either as a trigger for hypoxia-sensitive release or as a therapeutic target addressed by oxygen delivery and HIF-pathway modulation. These approaches should not be classified together: only systems in which low oxygen directly controls material activation are intrinsically stimuli-responsive. Oxygen-delivering or HIF-modulating materials regulate hypoxia-related pathways but do not necessarily sense hypoxia before acting.

Local pH Changes

Luminal pH, drainage-fluid pH, and interstitial pH within anastomotic tissue represent different compartments and should not be used interchangeably. The colonic lumen is generally mildly acidic to neutral, whereas postoperative tissue acidification may result from ischemia, lactate accumulation, inflammation, and impaired carbon dioxide clearance.^58,59 Direct measurements of interstitial pH at colorectal anastomoses, however, remain limited.

Systemic hypercapnia has been associated with impaired macrophage migration and activation and with an increased risk of AL.⁶⁰ Nevertheless, arterial acid–base measurements do not establish the pH experienced by a material placed on the anastomotic surface. Similarly, prospective studies have reported lower postoperative drainage-fluid pH in patients who subsequently developed AL.⁵⁸ Drainage-fluid pH may therefore serve as an early clinical marker, but it cannot be assumed to reflect either luminal or tissue-interstitial pH. Local acidification may also alter immune-cell function, protease activity, and matrix turnover, although the direction and magnitude of these effects depend on the affected cell type, enzyme, and pH range.

Mechanistic studies show that pH shifts regulate immune cells by changing intracellular pH. For instance, elevated extracellular potassium can alter intracellular pH, activating the AMPK/Nrf2 pathway. This reprograms macrophage metabolism and helps tissue regeneration.⁶¹ pH-responsive nanoparticles, like interleukin-12 (IL-12), adjust local pH and prompt macrophages to switch from an M2-like to an M1-like state, highlighting pH’s regulatory role.⁶² Acidification from excess CO₂ can suppress macrophage inflammatory gene expression via carbonic anhydrase II (CA II), impairing macrophage movement and activation.⁶⁰ This reduces immune cell entry, weakens repair, and raises the risk of poor healing.

For material design, pH is a useful trigger only when the activation range of the material overlaps the pH encountered at its implantation site. Acid-labile bonds and charge-reversible structures can couple reduced pH to degradation, structural change, or cargo release. However, spatially resolved measurements of anastomotic tissue pH are scarce, and drainage-fluid measurements do not define the local activation conditions.⁵⁸ It therefore remains uncertain whether acidification after colorectal surgery reaches the range required to activate many pH-responsive systems originally developed for tumors or intracellular compartments.

Redox Imbalance

ROS are generated early after anastomotic injury and have concentration- and time-dependent effects on repair. Transient, localized ROS production supports antimicrobial defense and redox-dependent cell migration and proliferation. In contrast, sustained ROS accumulation damages membrane lipids, proteins, and DNA, promotes apoptosis and endothelial dysfunction, and compromises ECM integrity.^43,63 ROS should therefore be regarded as both physiological mediators and potential drivers of tissue injury rather than uniformly harmful molecules.

Activated neutrophils and macrophages are major sources of postoperative ROS. During the respiratory burst, neutrophils generate superoxide, hydrogen peroxide, and other reactive species while also releasing granule contents and forming neutrophil extracellular traps (NETs).^64,65 Macrophage-derived reactive oxygen and nitrogen species (RONS) likewise contribute to antimicrobial defense and inflammatory signaling, with their effects depending on activation state and local concentration.²⁷ When ROS production exceeds local antioxidant capacity, oxidative injury can amplify inflammatory signaling and prolong immune-cell recruitment. Bacterial invasion may further sustain neutrophil activation and couple ROS production to protease-mediated matrix degradation.⁶⁶ This feedback between oxidative stress, inflammation, and protease activity can weaken the anastomotic tissue.

ROS can cleave defined oxidation-sensitive chemical groups, making them accessible triggers for material degradation or cargo release.⁴³ However, ROS is a non-binary trigger: early production contributes to host defense, whereas persistent accumulation impairs tissue repair. Responsive systems should therefore be calibrated to reduce sustained oxidative stress without abolishing transient antimicrobial and repair-associated signaling. Local delivery of exogenous reactive oxygen and nitrogen species for antimicrobial treatment represents a separate redox-modulating strategy and should not be classified as ROS-responsive unless endogenous ROS directly controls material activation.⁴⁸

Dysregulated Protease Activity

Protease activity is required for colorectal anastomotic healing because controlled ECM degradation permits cell migration, angiogenesis, and matrix turnover. MMPs therefore have physiological functions during repair. However, prolonged or excessive proteolysis accelerates collagen loss, reduces anastomotic strength, and contributes to AL.^66,67 The therapeutic target is abnormal protease activity rather than complete enzyme inhibition.

Proteases in the anastomotic microenvironment originate from both host cells and microorganisms. Neutrophils, macrophages, and fibroblasts release MMPs during inflammation and tissue remodeling. Microbial dysbiosis can increase proteolysis through bacterial collagenolytic enzymes or by inducing host MMP activity. For example, Fusobacterium nucleatum activates the epithelial cell–neutrophil axis and increases MMP2 and MMP9 expression and activity. Recruited neutrophils further release MMPs and neutrophil extracellular traps, promoting collagen degradation and loss of biomechanical strength.⁶⁶ Enterococcus faecalis can also enhance collagen degradation through activation of host MMP9, thereby increasing the risk of AL.⁶⁷

Proteases provide defined cleavage mechanisms for responsive material design, but elevated activity is not restricted to pathological healing. MMP2 and MMP9 also participate in physiological remodeling. Protease-sensitive substrates must therefore be evaluated for enzyme selectivity, activation range, cleavage kinetics, and compatibility with the healing stage. Materials that inhibit excessive proteolysis should also be distinguished from systems in which protease cleavage directly triggers degradation or cargo release.

Microbe-Derived Molecular Cues

Colorectal anastomoses are exposed to a dense luminal microbiota, and surgical disruption of the mucosal barrier increases contact between the wound, microorganisms, and microbe-associated molecular patterns (MAMPs).^68,69 These signals can alter healing through innate immune pathways in addition to microbial collagen degradation.⁶⁸ Their effects depend on ligand identity, abundance, barrier integrity, spatial exposure, and host receptor context rather than a simple commensal–pathogen distinction.

Individual microbial signals can produce different responses. In a colonic injury model, lipid-associated components of Akkermansia muciniphila activated MyD88-dependent epithelial signaling, increased IL-22 production, and improved wound repair.⁶⁹ By contrast, Fusobacterium nucleatum peptidoglycan activated epithelial NOD1/RIPK2/ERK signaling, increased IL-1β release and neutrophil recruitment, and promoted inflammation and collagen degradation. These findings illustrate the biological relevance of microbial ligands, although their effects in colorectal anastomoses require validation within the specific spatial and microbial context of the postoperative wound.

MAMPs present a specificity problem for responsive material design because lipopolysaccharide, peptidoglycan, and related ligands are abundant in the normal colonic lumen. Their presence alone may therefore trigger luminally exposed materials before leakage or pathological inflammation develops. Barrier breach, locally increased ligand concentration, bacterial enzyme activity, or combinations of microbial and host inflammatory cues may provide more selective activation conditions. Delivery of Toll-like receptor (TLR) or NOD-like receptor (NLR) antagonists can suppress downstream microbial signaling, but such systems should not be classified as MAMP-responsive unless ligand exposure directly controls material degradation, structural change, or cargo release.⁶⁸

Spatiotemporal Coupling of Anastomotic Cues

Anastomotic cues differ in magnitude, duration, and location. Hypoxia and ROS are prominent early after surgery, whereas persistent inflammation and microbial exposure can sustain pH disturbance, protease activity, and matrix degradation.^70,71 Their distribution also varies across the mucosa, submucosa, muscularis, and serosa because perfusion, microbial contact, and immune-cell infiltration are uneven.^72,73 A cue should therefore not be considered a useful material trigger solely because it is associated with anastomotic leakage. Its local concentration, exposure time, accessibility to the material, and overlap with physiological healing must also be defined. Single-cue systems may be affected by spatial and interpatient variability, particularly when the target signal also occurs during normal repair. Quantitative human data for these parameters remain limited, which constrains the selection of clinically relevant activation ranges.

Stimuli-Responsive and Microenvironment-Modulating Nanomaterial Strategies

Stimuli-responsive nanomaterials undergo defined structural or chemical changes when exposed to local cues, thereby coupling pH, ROS, protease activity, or hypoxia to material degradation, mechanical transformation, or cargo release. This section distinguishes cue-triggered systems from materials that modulate the same microenvironment without being directly activated by it (Figure 3). To maintain this mechanistic distinction while preserving application-specific evidence, Table 1 summarizes material platforms that have been directly evaluated in colorectal or colonic anastomotic models. Supplementary Table S1 separately summarizes responsive mechanisms demonstrated in gastrointestinal or non-anastomotic models that may inform future anastomosis-specific material design. Inclusion in Supplementary Table S1 therefore indicates mechanistic or translational relevance rather than demonstrated efficacy in colorectal anastomotic healing.

Figure 3.

Classes of cue-responsive materials and their distinction from microenvironment-modulating strategies relevant to colorectal anastomotic repair. Representative pH-, ROS-, enzyme-, and hypoxia-responsive systems are organized according to three linked elements: the biological trigger, the molecular switch, and the resulting material response. pH-responsive systems may use protonation–deprotonation or acid-labile bonds to regulate swelling, disassembly, degradation, or cargo release. ROS-responsive systems use oxidation-sensitive chemistries to couple elevated ROS to bond cleavage, carrier degradation, or release; ROS scavenging without ROS-dependent material transformation is classified as microenvironment modulation rather than intrinsic ROS responsiveness. Enzyme-responsive systems use cleavable substrates to couple local enzyme activity to degradation, structural change, or controlled release. Hypoxia-responsive systems use oxygen- or reductive-condition-sensitive chemical groups to produce material transformation or cargo release. By contrast, interventions such as HIF-pathway modulation or oxygen delivery alter the hypoxic microenvironment without necessarily being activated by hypoxia itself and are therefore distinguished as hypoxia-modulating strategies. Multi-stimuli platforms may integrate several responsive modules or logic-gated activation mechanisms. Solid outlines denote intrinsically cue-triggered material responses, whereas dashed outlines denote microenvironment-modulating strategies that do not require cue-dependent material activation. These mechanisms represent candidate signal-to-material response routes; their relevance to colorectal anastomotic repair depends on material-specific activation ranges, response kinetics, and direct experimental validation.

pH-Triggered Systems

pH-responsive materials convert changes in proton concentration into alterations in polymer ionization, bond stability, network structure, or cargo release.^74–77 Postoperative acidification has been proposed as a local trigger for anastomotic repair, but its suitability depends on whether the pH at the material–tissue interface reaches the activation range of the selected chemistry. Luminal pH, drainage-fluid pH, and tissue-interstitial pH represent different compartments and cannot be used interchangeably. Because spatially resolved measurements of anastomotic tissue pH remain limited, an association between lower pH and anastomotic leakage does not by itself establish that a pH-responsive material will activate in situ.

Two principal mechanisms are used. The first relies on protonation or deprotonation of ionizable groups. Carboxyl-containing polymers, including poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA), and amine-containing polymers, such as polyhistidine and poly(2-vinylpyridine), undergo changes in charge and hydrophilicity as pH varies.^78,79 These changes can alter hydrogel swelling, membrane permeability, or micelle and vesicle stability.⁷⁴ Such responses are usually continuous rather than binary and depend on the polymer pKa, ionic strength, and crosslink density. The second mechanism uses acid-labile bonds, including acetal, hydrazone, and imine linkages, to couple reduced pH to network degradation or cargo release.^80,81 Unlike reversible ionization, bond cleavage is generally irreversible; premature cleavage may therefore reduce mechanical support before the anastomosis has regained sufficient strength.

Chitosan is a representative protonatable polymer. Protonation of its amino groups increases hydration, swelling, and solubility, which can facilitate drug release under reduced pH.⁸² Chitosan also has antimicrobial and mucoadhesive properties, although their magnitude depends on molecular weight, degree of deacetylation, formulation, and environmental conditions.^83,84 Complete dissolution or rapid release may require a larger pH decrease than is present in extracellular anastomotic tissue.⁸⁵ Schiff-base hydrogels formed from chitosan and oxidized polysaccharides introduce acid-sensitive imine bonds and can provide self-healing, adhesion, and controlled release.^86,87 Their usefulness for anastomotic repair will depend on whether acid-triggered bond exchange or cleavage can occur without causing premature loss of network integrity.

Zeolitic imidazolate framework-8 (ZIF-8) provides a more sharply defined acid-triggered mechanism. It remains relatively stable near neutral pH but dissociates at approximately pH <6, releasing its cargo and zinc-containing components.^75,88 This activation range has been established mainly in tumors, lysosomes, or other strongly acidic compartments and may not be reached in extracellular anastomotic tissue. ZIF-8 therefore illustrates the need to compare material activation thresholds with measured local pH rather than assuming transferability from tumor models. Eudragit-based coatings use regional gastrointestinal pH to achieve colonic delivery.^89,90 They should be distinguished from pathology-responsive materials because their trigger is intestinal location rather than postoperative anastomotic acidification. Variation in gastrointestinal pH can still cause premature or delayed release, but this is a limitation of regional targeting rather than evidence for local disease-responsive activation.

For anastomotic applications, pH sensitivity should not be maximized without regard to specificity. The activation range must overlap the pH encountered at the intended implantation site while remaining sufficiently separated from physiological variation. Direct measurements of tissue-interstitial pH, exposure duration, and spatial gradients are therefore needed before pH-triggered systems can be rationally selected for colorectal anastomotic repair. Consistent with this limitation, none of the direct anastomosis studies summarized in Table 1 established a quantitative colorectal tissue-pH threshold that governed material activation. The pH-responsive systems summarized in Supplementary Table S1 should therefore be interpreted as transferable chemical design strategies rather than validated anastomosis-specific triggers.

ROS-Triggered and Redox-Modulating Systems

ROS, including superoxide, hydrogen peroxide, and hydroxyl radicals, accumulate when postoperative inflammatory activity exceeds local antioxidant capacity.^12,91 Two material strategies should be distinguished. In ROS-triggered systems, endogenous ROS directly controls bond cleavage, network degradation, hydrophilic transition, or cargo release. In redox-modulating systems, the material consumes or generates reactive species without necessarily undergoing ROS-dependent activation.

Thioketal linkers are widely used in ROS-triggered carriers and crosslinked networks. Oxidative cleavage disrupts the linker and promotes carrier degradation or cargo release.^92–94 Thioketal-containing polypeptide nanocarriers have shown H₂O₂ concentration-dependent release, whereas a thioketal-crosslinked hydrogel patch released CCL22 (C-C Motif Chemokine Ligand 22) in a diabetic wound model.^94,95 The latter provides evidence for the release mechanism outside an anastomosis model rather than direct support for colorectal repair. Thioketal responsiveness depends on linker structure, ROS species, exposure time, and material architecture; therefore, neither the cleavage products nor the activation threshold should be generalized across formulations.

Arylboronate linkers and dynamic boronate ester networks use related but distinct chemistries. In self-immolative arylboronate linkers, H₂O₂ oxidation can initiate subsequent bond fragmentation and payload release, including 1,6-elimination when an appropriate self-immolative spacer is present.⁹⁶ By contrast, dynamic boronate ester networks are formed through reversible interactions between boronic acids and diols. Oxidation can disrupt these crosslinks, while pH also affects bond formation, network swelling, and stability.^97,98 These mechanisms should not be described as a single reaction pathway. Arylboronate systems may respond rapidly to H₂O₂, but their kinetics depend on substituent structure and local pH.^99,100 In one hybrid design, ROS cleavage released dithiothreitol, which subsequently consumed residual ROS; this system therefore combined ROS-triggered degradation with redox modulation.⁹⁸

Thioether-containing polymers, represented by poly(propylene sulfide) (PPS), undergo stepwise oxidation to more hydrophilic sulfoxide and sulfone products. This transition can destabilize micelles or nanoparticles and release encapsulated agents.^101–103 Because thioether oxidation also consumes ROS, PPS systems can function as both ROS-triggered carriers and redox-modulating materials.^104,105 A PPS nanoparticle–hyaluronic acid hydrogel reduced oxidative damage to encapsulated cells and limited oxidative degradation of the hydrogel matrix.¹⁰¹ However, the ROS exposure required for a useful response and the tissue compatibility of oxidized products must be established for each formulation.

Catalytic antioxidant materials should be classified separately when ROS does not directly control material transformation. Selenium nanoparticles, for example, can exhibit glutathione peroxidase-like activity and reduce oxidative stress.¹⁰⁶ SeNP-containing hydrogels and microspheres have shown antioxidant effects in non-anastomotic tissue-repair or intestinal inflammation models.^106,107 These systems are redox-modulating rather than intrinsically ROS-triggered unless oxidation also governs network degradation or cargo release. Their application is further limited by dose-dependent toxicity and the possible formation of cytotoxic selenium oxidation products.¹⁰⁸ Materials that release exogenous RONS for antimicrobial treatment likewise represent redox modulation, not ROS responsiveness, unless endogenous ROS initiates their activation. Direct colorectal anastomosis studies further illustrate this distinction. Wang et al developed an in situ hydrogel composed of oxidized hyaluronic acid, adipic-acid-dihydrazide-modified hyaluronic acid, ε-poly-L-lysine, and tannic acid that combined tissue sealing with antibacterial and ROS-scavenging activity in rat and rabbit colon-resection anastomotic-leak models.⁴³ Because endogenous ROS did not control hydrogel formation or cargo release, this platform is more appropriately classified as redox-modulating rather than ROS-triggered. Similarly, the cold-atmospheric-plasma-activated hydrogel reported by Deng et al delivered exogenous reactive oxygen and nitrogen species to rat colonic anastomotic-leak sites and promoted antimicrobial, anti-inflammatory, and epithelial-reparative effects.⁴⁸ Its activity likewise represents controlled redox modulation rather than activation by endogenous postoperative ROS.

For colorectal anastomotic repair, these systems must balance three competing requirements: sensitivity to sustained pathological oxidative stress, preservation of early antimicrobial ROS signaling, and maintenance of mechanical support while tissue strength is restored. ROS are spatially restricted and often short-lived, while more stable species such as H₂O₂ may vary markedly with inflammation, perfusion, and bacterial burden.¹⁰⁹ Most reported systems have been evaluated in tumors, diabetic wounds, liver injury, or intestinal inflammation rather than colorectal anastomoses. Quantitative data on ROS concentration, duration, and distribution at human anastomotic surfaces remain limited. Material selection should therefore be based on a measured activation range and response time rather than on the presence of oxidative stress alone.

Protease-Triggered and Leakage-Activated Systems

Protease-sensitive nanomaterials incorporate cleavable peptide or polysaccharide substrates into carriers or crosslinked networks. Local enzymatic cleavage can then induce network degradation, structural disassembly, or cargo release.¹¹⁰ For colorectal anastomotic repair, three mechanisms should be distinguished: protease-triggered cleavage, enzyme-catalyzed in situ gelation, and leakage- or protein-activated reinforcement. Only the first is strictly pathology-responsive when endogenous protease activity directly controls material behavior.

Protease-cleavable systems commonly use substrates recognized by MMPs, whose activity may increase during inflammation and impaired anastomotic healing.^40,111 Peptide sequences such as GPLGIAGQ and CVPLSLYSG have been incorporated into hydrogel crosslinks to couple proteolysis to network degradation or cargo release.¹¹² A recent non-anastomotic example provides a more direct demonstration of enzyme-regulated therapeutic release. Meng et al constructed a hydrogel by crosslinking oxidized dextran, an MMP-9-sensitive peptide, and carboxymethyl chitosan containing M2-macrophage-derived exosomes. Elevated MMP-9 altered hydrogel degradation and exosome-release kinetics in a diabetic-wound setting.¹¹³ This study demonstrates formulation-specific MMP-9-responsive release, but it does not establish the MMP-9 activity range required for activation at a colorectal anastomosis. Peptide substrates provide greater molecular selectivity than bulk pH- or ROS-sensitive chemistries, but they are not exclusive to a single MMP. Cleavage may also be affected by related proteases, physiological MMP activity, substrate accessibility, crosslink density, pH, and endogenous inhibitors. Enzyme concentration alone therefore does not determine the degradation rate. Lysozyme-degradable chitosan microgels carrying pH-sensitive liposomes illustrate a cascade design in which enzymatic degradation releases a secondary carrier.¹¹⁴ However, the lysozyme concentration required for activation and its relevance to colorectal anastomotic tissue remain to be established.

Enzyme-catalyzed gelation uses an enzymatic reaction to form rather than cleave a network. Fibrin sealants, for example, use thrombin to convert fibrinogen into fibrin and permit rapid in situ filling and sealing.¹¹⁵ When thrombin is supplied as part of the formulation, the material does not sense pathological enzyme overexpression and should be classified as enzyme-catalyzed gelation rather than an endogenous enzyme-responsive system. Such materials provide immediate closure but do not inherently adjust their structure or release profile during later healing. Leakage-activated reinforcement represents a separate mechanism. In tannin-containing adhesives, leaked protein-rich fluids interact with tannins and increase network stiffness and tissue adhesion.¹¹⁶ The response is mediated by protein complexation rather than cleavage by a specific enzyme; it is therefore more accurately described as protein-mediated or leakage-activated reinforcement. This design may strengthen the seal when luminal contents contact the material, but its response depends on the composition and volume of the leaked fluid. Irreversible hardening, compatibility with digestive fluids, and subsequent tissue remodeling require further evaluation. Protease-triggered degradation may couple cargo release to local inflammation, but rapid early cleavage could remove mechanical support before sufficient collagen has formed. Network degradation must therefore be slow enough to preserve structural support during the period of lowest anastomotic strength. Because MMP2 and MMP9 also participate in physiological cell migration, angiogenesis, and matrix turnover, protease-sensitive systems should be evaluated for substrate selectivity, activation range, cleavage kinetics, retained mechanical strength, and interpatient variability.

Hypoxia-Sensing and Hypoxia-Modulating Systems

Nanomaterial strategies related to hypoxia can be divided into two groups (Figure 4). In hypoxia-sensing systems, reduced oxygen availability or associated reductive reactions directly control material disassembly, structural transition, or cargo release. In hypoxia-modulating systems, the material alters HIF signaling or oxygen availability without being activated by hypoxia. This distinction is necessary because pharmacological HIF stabilization and oxygen supplementation target hypoxia but are not inherently stimulus-responsive.

Figure 4.

Hypoxia-responsive, hypoxia-modulating, and hypoxia-conditioned strategies relevant to colorectal anastomotic healing. Anastomotic hypoxia may coexist with inflammation, ROS accumulation, and impaired vascularization, potentially contributing to a self-reinforcing cycle of impaired repair. The left panel depicts intrinsically hypoxia-responsive systems, in which low oxygen availability or an associated reductive environment directly initiates a chemical transformation, such as reduction or cleavage of hypoxia-sensitive groups, followed by material transformation or cargo release. Representative design concepts include azo- and nitroimidazole-based switches and other reduction-sensitive chemistries. The middle green panels depict hypoxia-modulating interventions, including HIF-pathway modulation and oxygen-generating or oxygen-delivering materials; these systems act on hypoxia-associated pathways or oxygen availability but should not be classified as hypoxia-responsive unless hypoxia itself controls their activation. Hypoxia-conditioned cells or extracellular vesicles are shown separately as conditioned regenerative cargo, because hypoxic preconditioning occurs before administration and does not constitute in situ hypoxia sensing by the material. Potential biological outcomes include improved oxygen availability, angiogenesis, control of inflammatory or oxidative injury, tissue regeneration, and preservation of anastomotic integrity. The illustrated strategies remain subject to formulation-specific activation, oxygen-delivery kinetics, safety, and validation under gastrointestinal anastomotic conditions.

Hypoxia-Responsive Molecular Switches

Azo linkages and nitroimidazole groups are commonly used as hypoxia-sensitive units. Azo bonds can be reduced and cleaved by azoreductases, leading to carrier degradation or cargo release.^117,118 In the colon, however, azo cleavage may also be driven by bacterial azoreductases, making it difficult to attribute material activation specifically to tissue hypoxia. The abundance and activity of these enzymes may also vary with microbial composition and luminal exposure.

Nitroimidazole groups undergo reduction under low-oxygen conditions, which can increase polymer hydrophilicity and destabilize micelles or nanoparticles.^119,120 This mechanism has mainly been evaluated in tumor models using drug-loaded carriers. Such tumors may contain more severe and sustained hypoxia than postoperative anastomotic tissue. The oxygen threshold and exposure time required for nitroimidazole reduction may therefore not match the milder or more transient hypoxia encountered at an anastomosis.¹²¹ Direct measurements are needed before these systems can be transferred to colorectal repair. Reduction-sensitive disulfide bonds should not be classified as hypoxia-specific switches. Their cleavage is primarily governed by reducing agents such as glutathione, which are abundant in intracellular environments and are not specific to low oxygen. Disulfide- and glutathione-responsive systems are therefore better classified as redox-responsive materials rather than hypoxia-responsive materials.^122,123

HIF-Pathway Modulation

HIF-pathway modulation targets the cellular response to hypoxia but does not require hypoxia to activate the material. Under normoxia, PHDs promote HIF-1α degradation; reduced PHD activity stabilizes HIF-1α and induces genes involved in metabolic adaptation and angiogenesis.⁵⁰ In murine colonic anastomosis models subjected to ischemic or infectious stress, PHD2 haploinsufficiency or pharmacological PHD inhibition increased bursting pressure, enhanced collagen deposition, and reduced leakage.⁵⁰ This study provides direct anastomotic evidence for HIF-pathway modulation, although it does not establish the efficacy of a specific nanomaterial carrier.

Local nanomaterial delivery has been used to reduce systemic exposure to HIF-modulating agents. For example, covalent incorporation of deferoxamine into a GelMA hydrogel prolonged local release and activated HIF-1α/VEGF signaling in a bone-repair model.¹²⁴ This result supports the feasibility of sustained local delivery but does not demonstrate anastomotic efficacy. Related systems containing dimethyloxalylglycine or cobalt ions have also been evaluated in non-anastomotic tissue-repair models.^125–129 Sustained HIF activation should not be judged solely by its angiogenic effects. PHD inhibition and iron chelation can affect other oxygen- and iron-dependent hydroxylation reactions, including processes required for collagen maturation. Excessive or prolonged HIF-1α stabilization may also promote fibrosis or support tumor-associated signaling.^130,131 These risks are relevant after colorectal cancer surgery. Local dose, release duration, tissue retention, and effects on collagen quality therefore require evaluation alongside vascularization. Cobalt-containing materials present the additional concern of dose-dependent ion toxicity.^130,131

Oxygen-Generating and Oxygen-Carrying Systems

Materials that increase local oxygen availability can be classified as oxygen-generating systems or oxygen carriers. Solid peroxides, including calcium peroxide (CaO₂), generate oxygen after contact with water but also produce hydrogen peroxide as an intermediate.^132–134 Catalase can be incorporated to convert hydrogen peroxide into water and oxygen and thereby reduce oxidative injury.^135–137 Because decomposition rates depend on particle size, encapsulation, water access, and local chemistry, oxygen output cannot be assumed to match postoperative tissue demand.

MnO₂ produces oxygen by catalysing the decomposition of endogenous hydrogen peroxide.^138,139 Its oxygen generation is therefore substrate-dependent: limited local hydrogen peroxide will restrict oxygen output, whereas high substrate concentrations may indicate substantial pre-existing oxidative stress. Acidic conditions may alter MnO₂ reaction rates, but the relationship between local pH, hydrogen peroxide concentration, oxygen production, and tissue toxicity must be measured for each formulation.

Perfluorocarbons (PFCs) do not generate oxygen. They physically dissolve and transport oxygen, and their effect depends on initial oxygen loading, local oxygen gradients, carrier retention, and gas exchange with surrounding tissue.^140,141 They should therefore be described as oxygen carriers rather than long-term oxygen reservoirs. Across these systems, the duration of oxygen supply varies widely, and it remains unclear whether reported release profiles match the intensity and duration of hypoxia at colorectal anastomoses. Excessive oxygenation may also increase oxidative stress, whereas inadequate delivery may not alter tissue oxygen tension.

Hypoxia-Conditioned Regenerative Cargos

Hypoxia-conditioned cells and extracellular vesicles represent an adjunct strategy rather than a hypoxia-responsive material class. In these systems, cells or vesicles are exposed to hypoxia before incorporation into a scaffold or hydrogel, thereby altering their cargo and regenerative activity before implantation. Hypoxia-conditioned adipose-derived stem-cell exosomes and engineered extracellular vesicles carrying stabilized HIF-1α have improved repair in non-anastomotic wound models.^142,143 However, the implanted construct does not necessarily sense local oxygen levels. These approaches should therefore be described as conditioned regenerative cargos. Their translation is limited by variable cargo composition, manufacturing scale, batch consistency, storage stability, and the absence of direct colorectal or colonic anastomosis evidence.

Overall, direct evidence for hypoxia-targeted nanomaterials in colorectal anastomoses remains limited. Azo and nitroimidazole systems require validation against measured anastomotic oxygen levels, while HIF-modulating and oxygen-supplying materials require assessment of dose, duration, collagen maturation, oxidative injury, and retained mechanical strength. Nitroimidazole derivatives and their reduction products also require formulation-specific biocompatibility testing.¹⁴⁴ Studies in gastrointestinal models should further account for bacterial enzymes, digestive fluids, microbial exposure, and peristaltic loading before conclusions are drawn from tumor, bone, skin, or urethral repair models.

Multi-Input and Sequentially Responsive Platforms

Single-cue systems may be affected by spatial, temporal, and interpatient variation, particularly when the selected signal also occurs during physiological healing. Multi-input materials combine two or more responsive elements to reduce this dependence on a single cue or to sequence different functions over time (Figure 5). Their activation logic must be specified. In an AND-gated system, all inputs must reach their respective activation ranges before the material responds, which can improve specificity but may delay or prevent activation when one signal is weak. An OR-gated system responds to any qualifying input, increasing sensitivity at the cost of nonspecific activation. Cascade systems instead use one input to expose, generate, or amplify a second responsive process. These designs are not inherently superior to single-cue systems and require validation of each input threshold, response order, and combined effect.

Figure 5.

Design logic and trade-offs of multi-input responsive materials for colorectal anastomotic repair. Candidate inputs include pH, ROS, protease activity, microbial activity, and hypoxia. The figure emphasizes how these cues can be combined within multi-input material architectures rather than repeating their individual biological mechanisms. Multifunctional integration incorporates several responsive or therapeutic components within one platform but does not necessarily demonstrate input-dependent logic and is therefore indicated by a dashed outline. True multi-input responsive systems, indicated by solid outlines, require one or more defined cues to control material activation. Sequential or cascade designs use an initial stimulus to expose, activate, or amplify a subsequent process. Representative combinations include pH/enzyme-, pH/ROS-, and pH/microbial-activity-responsive systems. AND-gated designs require all specified inputs to satisfy the activation condition and may improve specificity, whereas OR-gated designs can respond when one of several inputs is sufficient and may increase sensitivity at the expense of off-target activation. These design concepts should be evaluated according to individual and combined activation ranges, signal order, response latency, reversibility, retained mechanical function, and validation in colorectal anastomotic models. Solid outlines denote cue-dependent multi-input activation; dashed outlines denote multifunctional integration or systems in which explicit logic-gated activation has not been demonstrated.

Dual-input DNAzyme sensors illustrate the feasibility of AND-gated signal processing. In one electrochemiluminescent platform, signal output required both reduced pH and H₂O₂.¹⁴⁵ This study provides proof of concept for molecular logic but does not establish that the same architecture can function as an implantable therapeutic material at a colorectal anastomosis. Some pH/enzyme combinations have been developed for oral colon-targeted delivery. These systems use regional gastrointestinal pH together with degradation by microbial enzymes to reduce release in the upper gastrointestinal tract.^{32,89,90,146,147} They provide regional targeting rather than anastomosis-specific activation. A related distinction is supported by direct anastomosis evidence. Lee et al developed orally administered pectin/chitosan-coated polymeric nanoparticles carrying the inflammation-resolving peptide Ac2-26. The coating was designed to protect the particles during upper gastrointestinal transit and permit microbiota-mediated release in the colon, after which collagen-IV targeting promoted localization to injured intestinal tissue.⁴¹ In a murine model combining preoperative colitis with intestinal anastomosis, this platform improved macroscopic wound closure and histological healing. The system nevertheless represents microbiota-assisted regional delivery and wound targeting rather than activation by a quantitatively defined pathological microbial threshold. The same platform was subsequently evaluated using systemic, oral, and rectal delivery in a distal colo-colonic anastomosis model with acute Crohn’s-disease-like inflammation, further supporting its applicability to risk-enriched anastomotic conditions.⁴⁶ Moreover, pepsin and trypsin are upper gastrointestinal digestive enzymes and should not be grouped with microbial glycosidases as colon-specific inputs. Variation in luminal pH, microbial composition, and enzyme activity may still cause incomplete or premature release. pH/ROS-responsive networks more directly reflect inflammatory conditions in which local acidification and oxidative stress coexist.^148,149 However, the presence of both responsive groups does not demonstrate functional coupling. In some networks, pH changes bond stability, ionization, or polymer swelling and thereby alters the accessibility of ROS-sensitive groups.^150,151 This interaction must be demonstrated for each formulation rather than assumed. AND-gated activation may avoid release during mild physiological variation but fail when only one pathological signal reaches the required range. OR-gated activation may respond earlier but increases the probability of activation outside the intended site. Microbial metabolites and enzymes can provide additional inputs for antibacterial release.¹⁵² Short-chain fatty acids, lactate, β-glucuronidase, and related signals, however, are also produced by the normal colonic microbiota. Their high physiological background and interpatient variability limit infection specificity. These systems should not be described as pathogen-selective unless activation has been compared across commensal and pathogenic communities. Likewise, a response is self-limiting only when the material response is reversible and declining microbial activity has been shown to reduce release.

Multi-input systems therefore involve a trade-off among sensitivity, specificity, response time, and material complexity (Figure 5 and Supplementary Table S1). Additional responsive components may alter network mechanics, degradation, sterilization stability, and manufacturing reproducibility. For colorectal anastomotic repair, evaluation should include the individual and combined activation ranges, gate logic, response sequence, reversibility, retained mechanical strength, and direct validation in anastomosis models.

A Cue–Threshold–Response Framework for Anastomosis-Specific Nanomaterial Design

The presence of a disease-associated cue does not by itself justify its use as a material trigger. A useful trigger must be accessible at the implantation site, reach the activation range of the selected responsive chemistry, persist long enough to induce the intended material change, and remain distinguishable from physiological healing. The framework in Figure 6 therefore links cue identity and location, signal magnitude and duration, material activation range, physicochemical response, and intended therapeutic output. A distinction is required between the magnitude of a biological cue present at the anastomosis and the activation range measured for a material formulation. For several candidate cues, including interstitial pH, local ROS, protease activity, and oxygen tension, quantitative measurements at the colorectal anastomotic tissue–material interface remain limited. Accordingly, numerical activation conditions should be reported only when they were measured for the specific formulation, and these values should not be interpreted as colorectal physiological thresholds unless the corresponding cue was measured in the anastomotic compartment. Conditions derived from tumor tissue, intracellular compartments, drainage fluid, luminal contents, or non-anastomotic wound models cannot substitute for tissue-interstitial measurements. Where such measurements are unavailable, assigning an estimated numerical threshold would introduce unsupported precision.

Figure 6.

Signal–threshold–response framework for quantitative evaluation of responsive materials in colorectal anastomotic repair. The framework links four sequential levels of material design and evaluation: pathological signal, molecular recognition, material transformation, and biological or mechanical outcome. Candidate inputs include hypoxia, local pH changes, ROS, protease activity, and microbe-derived signals. Quantitative implementation requires characterization of the magnitude or activity and exposure duration of the biological cue, followed by determination of the formulation-specific activation range or threshold, the response latency and release or degradation kinetics, and the resulting mechanical or biological endpoint. Responsive molecular switches may include hypoxia-reducible groups, acid-sensitive moieties, ROS-cleavable linkages, enzyme-cleavable substrates, and microbe-responsive components. Their activation can produce cargo release, swelling, network degradation, conformational changes, or surface-charge reversal, with downstream effects on angiogenesis, inflammatory regulation, antimicrobial activity, or mechanical support. Solid pathway lines denote cue–response relationships experimentally demonstrated in the original experimental system, whereas dashed lines denote chemically feasible or transferable pathways for which the complete relationship has not been directly demonstrated in the relevant setting. Experimental demonstration in another model should not be interpreted as validation in colorectal anastomotic tissue. The illustrative pathways therefore distinguish mechanistic evidence from anastomosis-specific validation and should be interpreted together with the evidence levels summarized in the accompanying tables.

The contribution of the present framework lies not in introducing new responsive chemistries or individual evaluation parameters, but in organizing them into an anastomosis-specific quantitative verification sequence. In practical terms, implementation requires four linked measurements: (i) the concentration or activity and postoperative time course of the selected cue at the intended material–tissue interface; (ii) the formulation-specific activation threshold or response range; (iii) the exposure duration required for activation and the subsequent response latency or kinetics; and (iv) the resulting material and therapeutic outputs. A candidate system would be considered appropriately calibrated when the pathological cue overlaps the material activation range for sufficient time to generate the intended response within the relevant therapeutic window, while preserving the mechanical support required at that stage of healing. Therapeutic output should then be assessed using prespecified endpoints appropriate to the platform, such as cargo release, wet-tissue adhesion or bursting pressure, leakage incidence, collagen organization, or inflammatory and microbial outcomes. Because quantitative human measurements at the anastomotic material–tissue interface remain limited, this framework should currently be regarded as an experimental calibration and reporting workflow rather than a validated clinical decision rule.

Candidate microenvironmental cues include reduced oxygen availability, local pH changes, ROS, elevated or sustained protease activity, and microbe-derived metabolites or enzymes. A cue becomes pathologically informative only when its magnitude, duration, or spatial distribution differs sufficiently from that observed during normal repair. Material activation is determined not only by the chemistry of the responsive unit but also by its presentation within the network. Accessibility, crosslink density, material thickness, molecular diffusion, ionic strength, competing substrates, and exposure time can all alter sensitivity and selectivity. Once activated, the material may swell, degrade, disassemble, change conformation, or alter surface charge. These changes can regulate cargo release or mechanical properties, but they do not directly guarantee anti-inflammatory, antibacterial, angiogenic, or reinforcing effects. Such outputs also depend on payload dose, bioactivity, retention, release duration, and the response of surrounding cells and microorganisms.

Threshold and timing mismatches can limit otherwise valid material chemistries. ZIF-8 provides a defined acid-triggered disassembly mechanism, but its typical activation below approximately pH 6 may not match the extracellular pH at a colorectal anastomosis.^75,88 Conversely, MMP-cleavable networks can couple protease activity to degradation or cargo release, but rapid cleavage during early inflammation may remove mechanical support before sufficient collagen has formed.^40,111,112 The nitroimidazole- and arylboronate-based pathways illustrated in Figure 6 likewise represent chemical design concepts rather than direct evidence of efficacy in colorectal anastomosis models.^96,119 Multi-input systems can combine several cue–response relationships, but greater complexity may introduce delayed activation, cross-reactivity, altered mechanics, or manufacturing variability.

Evaluation should therefore proceed from signal accessibility to activation-range matching, temporal compatibility, retained material function, and level of evidence. Luminal pH cannot be assumed to represent tissue-interstitial pH; short-lived ROS may not diffuse through the entire material; proteases may be matrix-bound or inhibited; and microbial signals may already be abundant in the normal colonic lumen. Responses that cause degradation or swelling should also be tested for their effects on adhesion, bursting pressure, resistance to peristaltic loading, and later tissue compliance. This framework does not replace structure-based material design. Rather, it adds the requirement that activation range, response kinetics, and functional output be matched to measured local cues. Its purpose is to distinguish materials that are responsive in principle from those likely to respond at the appropriate location and stage of anastomotic healing.

Design Requirements and Translational Constraints for Anastomosis-Specific Responsive Nanomaterials

The cue–threshold–response framework establishes whether a material can be activated at an anastomotic site. Effective systems must also retain wet-tissue adhesion and structural support, avoid suppressing physiological inflammation or matrix turnover, and remain compatible with manufacturing and surgical use (Figure 7). This section translates the framework into four design requirements: calibrated cue selection, integration of responsive chemistry with material mechanics, preservation of host and microbial homeostasis, and temporal coordination. Manufacturing, surgical deployment, and preclinical validation are then considered as constraints on material development.

Figure 7.

Design requirements and translational constraints for anastomosis-specific responsive nanomaterials. The proposed design workflow integrates four interdependent requirements. Pathological signal mapping identifies candidate local cues and determines whether their magnitude and duration enter a material-relevant activation window. Modular molecular responsiveness links selected cues to formulation-specific responsive chemistries while retaining control over assembly and response kinetics. Ecological function coupling considers inflammatory regulation, immune-cell behavior, extracellular-matrix remodeling, microbiota–material interactions, and the coordination of biological repair with mechanical support. Temporal synchronization aligns therapeutic or material functions with the overlapping inflammatory, proliferative, and remodeling phases and may include sequential or cascade activation where experimentally justified. Translation additionally requires scalable and reproducible manufacturing, sterilization compatibility, biosafety of the intact material and degradation products, surgical usability, and an appropriate regulatory pathway. These elements represent design and validation requirements rather than demonstrated clinical efficacy.

Cue Selection and Response Calibration

Candidate cues should be selected from measurements obtained at the intended material–tissue interface rather than from general associations with anastomotic leakage. Potential inputs include local pH, oxygen tension, ROS, protease activity, and microbe-derived metabolites or enzymes.^{40,50,51,66–69,90,91,111} For each cue, its anatomical compartment, concentration or activity range, postoperative time course, and interpatient variability should be defined. Luminal, drainage-fluid, and tissue-interstitial measurements should not be treated as equivalent. When a primary study does not report a quantitative activation range or response time, these parameters should remain explicitly unassigned rather than inferred from a chemically similar formulation. Likewise, an activation threshold is not applicable to constitutively active microenvironment-modulating platforms whose therapeutic action does not require an endogenous cue. This reporting distinction is used in Table 1 and Supplementary Table S1.

The material activation range should overlap the sustained pathological range while remaining sufficiently separated from physiological healing. Because many systems show graded swelling, oxidation, degradation, or release rather than binary switching, response onset, slope, latency, reversibility, and maximum response should be reported instead of a single nominal threshold. These properties can be adjusted through acid-labile bond stability, oxidation-sensitive linker chemistry, protease-cleavable substrate design, crosslink density, and transport through the material network.^{40,80,92,93,111} Rapid or complete activation is not always desirable: it may cause burst release or premature loss of mechanical support. A steep response is useful when physiological and pathological signal ranges are clearly separated, whereas a graded response may be safer when the cue changes continuously. Combined or sequential inputs may improve discrimination, but each input and their combination must be tested separately to determine the actual activation logic.¹¹⁴

Integrating Responsive Chemistry with Wet-Tissue Mechanics

Responsive chemistry should be incorporated without compromising adhesion, structural stability, or conformity to intestinal tissue. Acid-labile bonds, oxidation-sensitive linkers, and protease-cleavable substrates can regulate degradation and release, but their incorporation also changes crosslink density, swelling, molecular transport, and network strength.^{40,80,86,87,94,96,97,101,102,111,112} Their performance must therefore be assessed in the final material rather than inferred from the isolated responsive group. Combining several modules may further alter gelation time, network homogeneity, and mechanical reproducibility; modular components cannot be assumed to be interchangeable.

Recent direct anastomosis studies illustrate the importance of integrating biological activity with intestinal mechanical requirements. Ju et al developed a temperature-sensitive extracellular-matrix hydrogel based on porcine small-intestinal-submucosa ECM modified with boric acid and 4-ARM-PEG-SC. In a rat colonic anastomotic-leak model, the material combined anastomotic sealing with resistance to digestive-fluid exposure, tissue adhesion, antibacterial activity, and modulation of the local inflammatory and oxidative environment.⁴⁴ Zhu et al reported a γ-PGA-Cys-DA/oxidized-konjac-glucomannan/tannic-acid hydrogel in which dynamic thiol–aldehyde crosslinking produced gelation in approximately 12s together with wet-tissue adhesion, antibacterial activity, angiogenic effects, and improved healing in a rat colorectal anastomosis model.⁴⁵ Neither platform, however, should be interpreted as evidence that a quantified endogenous anastomotic cue triggered therapeutic activation; their direct evidence primarily supports deployment, sealing, and microenvironment modulation.

Therapeutic release and bulk structural degradation should be decoupled when early signal-triggered delivery is required but mechanical reinforcement must be retained. Cargo may be released through cleavable side chains, responsive particles, or a degradable secondary network while the primary adhesive network remains intact. This approach avoids the conflict between high early ROS or protease activity and the need for support during the period of lowest anastomotic strength. Swelling must also be limited because excessive fluid uptake can weaken adhesion, increase tissue compression, and promote delamination, although controlled swelling may facilitate molecular transport and release.^153,154

Mechanical testing should reflect the intended surgical setting. In addition to elastic modulus and tensile strength, evaluation should include wet-tissue adhesion, bursting pressure, cyclic loading that approximates peristalsis, stability in mucus and intestinal fluids, compatibility with sutures or staples, and resistance to delamination. Material persistence should be defined relative to the recovery of tissue strength rather than assigned uniformly to a healing phase. Premature degradation may expose the defect before sufficient collagen has formed, whereas prolonged stiffness or swelling may impair remodeling, narrow the lumen, or increase local inflammation. The required response profile therefore depends on whether the material functions primarily as a sealant, drug carrier, reinforcing patch, or combined system.

Preserving Physiological Repair and Microbial Homeostasis

Responsive materials should limit sustained inflammatory injury without suppressing the early host responses required for bacterial clearance and wound signaling. Transient reactive oxygen species production, immune-cell recruitment, and protease activity contribute to antimicrobial defense, debris removal, angiogenesis, and matrix turnover. Anti-inflammatory or antioxidant functions should therefore respond preferentially to excessive or persistent activity rather than to basal signals present during normal repair. The activation range must be established experimentally because quantitative boundaries between physiological and pathological inflammation at human anastomoses remain poorly defined. Macrophage responses should not be described as a uniform transition from an M1 to an M2 phenotype. Macrophages occupy heterogeneous, context-dependent states, and both prolonged inflammatory activation and excessive reparative signaling may impair healing. In murine colonic anastomosis models, PHD inhibition improved tissue repair and was associated with changes in macrophage function.⁵⁰ Similar immunomodulatory effects reported in non-anastomotic scaffold models¹²⁶ provide indirect evidence only. Material evaluation should therefore include inflammatory mediators, macrophage functions, bacterial burden, collagen organization, and tissue strength rather than relying solely on M1/M2 markers.

Antimicrobial activity must also be balanced against preservation of commensal functions. Sustained broad-spectrum release may disrupt microbial community structure, reduce colonization resistance, and select for antimicrobial resistance. Microbial metabolites and enzymes are not inherently pathogen-specific because many are present in the normal colonic lumen.¹⁵² More selective activation may require barrier disruption, locally increased bacterial enzyme activity, or combinations of microbial and host inflammatory cues. Evaluation should include biofilm burden, commensal composition, mucosal barrier recovery, and resistance selection. A system should be described as self-limiting only when declining microbial activity has been shown to reduce material activation or drug release.

Temporal and Sequential Programming

Anastomotic healing proceeds through overlapping inflammatory, proliferative, and remodeling phases rather than fixed, clearly separated intervals. Material behavior should therefore not be programmed according to a universal postoperative schedule. The timing of infection, ischemia, protease activity, angiogenesis, and matrix deposition varies with anastomotic location, perfusion, microbial exposure, and patient condition.

Temporal control can follow two complementary strategies. Time-programmed systems use predetermined changes in layer dissolution, diffusion, or network degradation to sequence material functions. Event-driven systems respond to local cues such as sustained ROS, protease activity, or microbial enzymes. The former provides predictable structural persistence but may become misaligned when healing is delayed; the latter adapts to local abnormalities but depends on reliable signal accessibility and activation-range matching. Hybrid systems may therefore retain a stable load-bearing network while using cue-responsive particles, side chains, or secondary networks for therapeutic release.

Sequential designs should be evaluated by the order, delay, and duration of each response rather than by the number of incorporated modules. Early antimicrobial or inflammation-modulating release, later support for vascularization, and subsequent material degradation represent possible sequences, not universal requirements. Each sequence must be compared with single-stage controls and tested for premature activation, incomplete transition, payload interference, and loss of mechanical support.

Manufacturing, Surgical Deployment, and Preclinical Translation

Clinical translation requires control of material composition and performance across manufacturing batches. Relevant quality attributes include polymer molecular weight, degree of functionalization, responsive-linker content, crosslink density, residual solvents or catalysts, payload loading, activation profile, wet adhesion, mechanical strength, and degradation kinetics. Multi-input materials should be tested under each input separately and in combination to confirm their response logic and exclude unintended cross-activation. Manufacturing specifications should therefore include functional response curves rather than chemical composition alone.

Sterilization and storage may alter responsive bonds, network structure, or payload activity. Autoclaving, ethylene oxide, irradiation, and aseptic processing should be selected according to material chemistry and formulation. Activation range, release kinetics, adhesion, swelling, and mechanical properties must be re-evaluated after sterilization and at the end of the proposed shelf life. Biosafety assessment should include the intact material, residual reagents, released drugs, degradation intermediates, and systemically absorbed products. Particular attention is required for oxidation products, low-molecular-weight cleavage fragments, selenium-containing components, nitroimidazole derivatives, and sustained HIF-pathway activation.^108,109,144 Local outcomes should include inflammation, peritoneal adhesion, luminal narrowing, delayed tissue injury, microbiota disruption, and antimicrobial-resistance selection.

Surgical usability should be assessed under clinically relevant conditions. Application time, mixing and curing requirements, repositionability, visibility, and compatibility with hand-sewn and stapled anastomoses should be reported. Materials intended for spraying, injection, or circumferential wrapping must adhere to bleeding or mucus-covered tissue, conform to low pelvic anatomy, and avoid interference with staples, sutures, or luminal patency. Preclinical testing should progress from intestinal-fluid exposure and cyclic mechanical loading to contaminated or ischemic anastomosis models. Studies should report leakage incidence, bursting pressure, abscess formation, adhesion, stenosis, collagen organization, degradation, and systemic toxicity, with comparisons against standard closure, non-responsive materials, or established sealants. Large-animal studies should reproduce clinically relevant deployment and anastomotic geometry before human evaluation. Regulatory classification will depend on the intended jurisdiction, composition, and primary mode of action; scaffold–drug systems may require coordinated device and pharmaceutical assessment, including manufacturing controls, release specifications, and pharmacological safety.

Discussion

Responsive nanomaterials can couple local cues to degradation, cargo release, or changes in physical properties. Their clinical value, however, depends on activation under conditions present at a colorectal anastomosis and on demonstrable benefit over simpler non-responsive materials. Most available evidence remains preclinical, and many systems were developed in tumors, skin wounds, bone defects, or intestinal inflammation. The discussion therefore focuses on evidence quality, proof of responsiveness and comparative benefit, and the clinical settings in which added material complexity may be justified.

Evidence Quality and Model Relevance

Evidence should be separated according to its proximity to the intended application. In this review, evidence quality is considered along two complementary axes: model relevance and mechanistic completeness. Model relevance reflects how closely the experimental setting reproduces colorectal anastomotic repair, whereas mechanistic completeness reflects whether the proposed cue was shown to activate the final material under quantified conditions and whether this activation was linked to a relevant material or biological outcome. These dimensions are considered separately because a directly tested anastomotic material may lack a defined cue–activation relationship, whereas a chemically well-characterized responsive system may have been evaluated only in a non-anastomotic model. Direct preclinical material evidence now includes collagen-IV-targeted Ac2-26 nanoparticle systems evaluated in inflamed intestinal or colonic anastomosis models,^41,46 a thermoresponsive extracellular-vesicle delivery hydrogel evaluated after colorectal irradiation,⁴² and multifunctional hydrogel sealants or barriers evaluated directly in colonic or colorectal anastomoses.^43–45,48 These studies demonstrate local delivery, mechanical sealing, physical responsiveness, or microenvironment modulation under anastomosis-specific conditions. However, their material functions were generally not initiated by a quantitatively defined endogenous pathological cue. Gastrointestinal but non-anastomotic models can clarify mucosal compatibility or microbial interactions, whereas tumor, skin, bone, and other tissue-repair models mainly demonstrate chemical responsiveness or general biological activity. Results from these settings should not be presented as evidence that a material will activate or improve healing at a colorectal anastomosis.

Standard healthy-rodent models may also overestimate efficacy. Young animals generally do not reproduce the impaired perfusion, altered microbiota, comorbid disease, nutritional deficiency, or previous chemoradiotherapy encountered in high-risk surgical patients.^43,50,155 Material performance may change when hypoxia, protease activity, bacterial exposure, and tissue-repair capacity differ in magnitude or duration. Models should therefore include defined risk conditions rather than rely only on technically uncomplicated anastomoses. Human data are particularly limited for the local signals used to activate these materials. Systemic risk factors, drainage-fluid measurements, and isolated tissue samples do not define the concentration, duration, or spatial distribution of cues at the material–tissue interface.^58,67 Large-animal studies should therefore follow quantitative signal characterization and reproduce clinically relevant features, including low pelvic anatomy, stapled or hand-sewn construction, impaired perfusion, luminal contamination, perioperative antibiotics, and laparoscopic or robotic deployment. These models should be used for preclinical validation rather than treated as substitutes for clinical evidence.

On the basis of the evidence reviewed here, the most defensible near-term direction is a relatively simple anastomosis-deployable platform in which one quantitatively characterizable responsive function is combined with mechanical support that does not depend on the same trigger for structural integrity. Such designs would allow cue-dependent release or modulation to be evaluated independently from sealing and reinforcement. In contrast, multi-input AND/OR-gated systems, sequential or cascade-responsive constructs, and materials whose activation conditions have been established only in tumor or other non-anastomotic environments remain largely proof-of-concept for colorectal anastomotic repair. Likewise, pH-, ROS-, protease-, or hypoxia-responsive chemistries should not be considered application-ready solely because their molecular response is well characterized in another model. Progress toward translation requires demonstration that the relevant cue is present at the material–tissue interface, that its magnitude and duration overlap the formulation-specific activation range, that activation produces a reproducible functional advantage over an otherwise comparable non-responsive system, and that this advantage is retained without compromising anastomotic mechanics or safety.

Demonstrating Responsiveness and Comparative Benefit

The incorporation of a responsive moiety does not establish that the finished material will activate at a colorectal anastomosis. Responsiveness should be demonstrated through concentration- and time-dependent testing within the signal range measured at the intended material–tissue interface, together with exposure to relevant competing cues. Many pH-, ROS-, protease-, and hypoxia-sensitive systems have been evaluated under marked acidification, high concentrations of exogenous H₂O₂, purified enzymes, or severe tumor hypoxia.^{40,75,88,94–98,111,112,117–120} These experiments establish chemical feasibility but do not confirm activation under postoperative anastomotic conditions. Multi-input systems also require separate testing of each input and their combination to determine whether the material follows AND, OR, sequential, or nonspecific coupled behavior.^148–151 This evidence gap is apparent when Table 1 is compared with Supplementary Table S1. Direct colorectal anastomosis studies provide clinically relevant information on implantation, sealing, local retention, and healing outcomes, but quantitative endogenous cue–activation relationships are seldom established. Conversely, many transferable systems have experimentally defined pH-, ROS-, enzyme-, or hypoxia-dependent responses, but these activation conditions were obtained outside the colorectal anastomotic environment. Neither evidence type alone establishes an anastomosis-specific cue–threshold–response relationship.

Stimulus responsiveness also introduces failure modes that are less prominent in passive materials. If activation occurs at physiological cue levels or too early after surgery, cleavage, swelling, or network destabilization may reduce crosslink density, wet adhesion, or load-bearing capacity before sufficient tissue strength has been restored.¹⁵⁶ Rapid carrier disassembly may produce burst release and unnecessarily high local drug exposure, whereas excessive degradation can shorten material residence and generate degradation products whose local or systemic compatibility has not been established.¹⁵⁷ Conversely, an activation range set above the signals actually present at the anastomosis may result in little or no therapeutic response. Biological interference is an additional concern. Excessive scavenging of ROS may suppress antimicrobial signaling, excessive inhibition of protease activity may interfere with physiological matrix turnover, and prolonged suppression of inflammatory pathways may delay the transition between healing phases. Responsive systems should therefore be evaluated for both premature and insufficient activation, and the consequences of activation should be assessed together with retained mechanical function and physiological repair rather than by release behavior alone.

The colorectal environment imposes additional biological, chemical, and mechanical stresses that may substantially alter material behavior compared with simplified in vitro conditions. Fecal contamination and the high local microbial burden can expose materials to bacterial enzymes, metabolites, and biofilm-associated activity that may accelerate degradation, alter release kinetics, or activate susceptible chemistries independently of the intended pathological cue.¹⁵⁸ Mucus and intestinal fluid can dilute soluble signals, restrict molecular transport to the material surface, promote swelling or leaching, and reduce adhesion or local retention. Luminal pH also varies with anatomical location, diet, microbial metabolism, and intestinal contents and should not be assumed to represent the pH at the tissue–material interface.¹⁵⁹ Digestive enzymes may further cleave peptide-, protein-, or polysaccharide-containing components independently of the designed responsive mechanism.³⁶ In parallel, peristaltic motion exposes anastomotic materials to repeated compression, shear, tension, and bending, while continuous intestinal deformation can fatigue both the material network and the tissue–material interface.^36,45 These factors may shift the apparent activation range, shorten residence time, alter release or degradation kinetics, and reduce retained mechanical support. Accordingly, candidate responsive systems should be evaluated under combined colorectal-relevant conditions, including mucus and intestinal-fluid exposure, microbial or fecal contamination where appropriate, variable pH and enzyme exposure, and cyclic mechanical loading. Such testing should assess cue-dependent activation together with adhesion, swelling, mass loss, local retention, release kinetics, and retained mechanical strength rather than evaluating responsiveness in isolated buffer systems alone.

Comparative controls are required to determine whether responsiveness contributes to efficacy. A responsive formulation should be compared with a composition-matched non-responsive analogue, an unloaded material, and the same payload delivered freely or through a non-responsive release system. Comparisons with established sealants, reinforcement materials, or standard anastomotic closure are also needed.^115,116 Without these controls, improved healing cannot be attributed to stimulus-dependent activation rather than to the payload, carrier, or mechanical coverage alone. Such comparisons are particularly important because non-responsive materials may themselves provide substantial anastomotic benefit. For example, Antonelli et al reported that an acellular urinary-bladder-matrix ECM hydrogel reduced leakage from 46% in untreated controls to 8% in a rat low-rectal-anastomosis model.⁴⁷ Although this platform is not stimulus-responsive, it provides a relevant benchmark against which the added complexity of responsive systems should be evaluated.

Evaluation should include activation range, response latency, release or degradation kinetics, wet adhesion, bursting pressure, cyclic mechanical stability, swelling, and retained strength after activation. Biological outcomes should include leakage incidence, abscess formation, peritoneal adhesion, stenosis, collagen organization, microbial effects, and systemic toxicity. Accordingly, Table 1 and Supplementary Table S1 summarize these parameters using the study-specific metrics reported in the original publications wherever data are available; parameters that were not quantitatively reported are left unassigned rather than extrapolated across non-equivalent formulations or experimental models. The additional manufacturing and regulatory complexity of a responsive system is justified only when it provides a measurable advantage in efficacy, safety, dose control, or mechanical performance over simpler alternatives.

Clinical Indications and Research Priorities

Responsive nanomaterials are unlikely to provide equal benefit across all colorectal anastomoses. Their added complexity may be most justified in settings with a high risk of impaired healing, including low rectal anastomoses, compromised perfusion, substantial luminal contamination, diabetes, malnutrition, or previous chemoradiotherapy.^5,6,155 Material selection should therefore be indication- and risk-stratified rather than described as fully personalized. Clinical risk factors may identify patients who require additional protection, but they do not establish that a specific local cue, such as low pH, elevated ROS, or protease activity, will reach the activation range of a material.

Translation will also depend on whether responsive behavior can be manufactured and maintained reproducibly outside small-scale laboratory preparation. Batch-to-batch control should include material composition, degree of functionalization, crosslink density, particle or network characteristics, payload loading, and the activation and release profile of the final formulation. Sterilization must be shown not to alter responsive groups, network structure, drug stability, or mechanical performance,¹⁶⁰ and storage studies should determine whether the activation range, gelation behavior, adhesion, and payload integrity remain within predefined specifications over the intended shelf life. Degradation products should be identified and evaluated separately from the intact material because stimulus-triggered cleavage can generate products with different local exposure and toxicity profiles. Surgical translation further requires reproducible delivery to wet or mucus-covered tissue, adequate working and curing times, compatibility with stapled or hand-sewn anastomoses, and retention under luminal fluid exposure and cyclic intestinal motion. These requirements become more difficult as additional responsive modules are introduced because each component adds manufacturing variables, release specifications, potential degradation products, and regulatory questions concerning the primary mode of action.¹⁵⁷ Multi-responsive systems should therefore be advanced only when direct head-to-head testing demonstrates a clinically relevant advantage in efficacy, safety, dose control, or mechanical performance over a simpler formulation.

Research priorities should follow a staged pathway. Candidate systems should first be tested in risk-enriched anastomosis models that reproduce ischemia, contamination, delayed healing, or low pelvic deployment. Only materials that retain adhesion and mechanical support while demonstrating cue-dependent benefit over non-responsive comparators should progress to large-animal evaluation. Early clinical studies should focus on surgical handling, deployment time, device compatibility, local retention, degradation, and short-term safety before attempting to demonstrate a reduction in anastomotic leakage.

Near-term priorities are therefore to quantify human anastomotic cues, define clinically relevant activation ranges, identify a limited number of high-risk indications, and establish standardized material and biological endpoints. Multi-input or sequential systems should be advanced only when they outperform simpler designs under the same conditions. Risk-stratified use is more realistic than manufacturing a different material for each patient.

Conclusion

Colorectal anastomotic repair requires both structural support and control of local biological disturbances. Stimuli-responsive nanomaterials can couple pH, ROS, protease activity, hypoxia, or microbe-derived cues to material transformation or cargo release. Direct colorectal anastomosis studies are now available for several targeted nanoparticle and hydrogel platforms, but this evidence remains preclinical and is derived predominantly from animal models. Importantly, many directly tested platforms function through targeted delivery, physical sealing, or constitutive microenvironment modulation rather than activation by a quantitatively defined endogenous anastomotic cue. Conversely, many chemically defined cue-responsive systems remain supported by non-anastomotic models. A staged research pathway is therefore required. First, human colorectal anastomotic cues should be quantified at the intended material–tissue interface, including their magnitude, duration, spatial distribution, and interpatient variability. Second, formulation-specific activation ranges and response kinetics should be validated under realistic gastrointestinal conditions that reproduce relevant pH, oxygenation, oxidative stress, protease activity, microbial exposure, and luminal fluids rather than relying on trigger conditions established in unrelated disease models. Third, responsive formulations should undergo wet-tissue adhesion, bursting-pressure, cyclic mechanical-loading, degradation, and retained-strength testing before and after activation, together with direct comparisons against composition-matched non-responsive controls and established closure or reinforcement materials. Finally, systems that demonstrate a reproducible cue-dependent advantage without loss of mechanical function or safety should be evaluated in clinically relevant large-animal models that reproduce anastomotic geometry, impaired perfusion, contamination, and surgical deployment. Manufacturing reproducibility and clinically relevant safety endpoints should be incorporated throughout this pathway. Until these requirements are met, stimulus-responsive nanomaterials should be regarded as experimental adjuncts rather than established alternatives for preventing or treating colorectal anastomotic leakage.

Funding Statement

This work was supported by the International Science and Technology Cooperation Project of Jilin Provincial Department of Science and Technology (20230402015GH).

Data Sharing Statement

No data was used for the research described in the article.

Author Contributions

All authors made a significant contribution to the work reported, whether that is in the conception, study design, execution, acquisition of data, analysis and interpretation, or in all these areas; took part in drafting, revising or critically reviewing the article; gave final approval of the version to be published; have agreed on the journal to which the article has been submitted; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.