Immune‐Responsive Bioinks Enable Vision‐Guided in Situ Robotic Bioprinting to Accelerate Diabetic Wound Healing

Background

Chronic diabetic wounds combine hypoxia, impaired angiogenesis and persistent pro-inflammatory M1 macrophages that fail to transition to pro-regenerative M2 states. Calcium peroxide (CaO2)-based oxygen-releasing materials can relieve local hypoxia, but they often show burst release, uncontrolled reactive oxygen species (ROS) generation and poor conformity to irregular wounds. Injected hMSCs act as a source of paracrine regenerative factors, yet they are poorly retained and lose viability in ischemic tissue. Most wound patches are prefabricated and mechanically static, which limits their fit to heterogeneous wound geometries.

Design and approach

CaO2 nanoparticles are encapsulated in zein, a hydrophobic plant protein, by antisolvent precipitation and coated with Pluronic F-127 to form zein-based oxygen-generating microparticles (zOMPs). The zein shell restricts water penetration and is intended to slow oxygen generation. The zOMPs are mixed, rather than encapsulated, with GelMA/gelatin colloidal particles made by water-in-water emulsion with poly(ethylene oxide), and hydrogen bonding between the particle surfaces gives a shear-thinning, photocrosslinkable bioink (Coll-zOMP); hMSCs are added to form Coll-zOMP-hMSC. The bioink is deposited by INSIGHT (Intelligent in situ bioprinting Guided by Eye-in-Hand robot Technology), which uses a six-degree-of-freedom robotic arm, automated wound recognition, point-cloud-based path generation and real-time visual feedback. The yellow color from zein also helps the camera recognize each printed layer.

How it was evaluated

The hydrogels were characterized by dynamic light scattering, SEM, AFM nanoindentation, rheology, swelling, collagenase degradation and oxygen and H2O2 measurement. Printability was tested by temperature- and shear-dependent viscosity, hanging filament tests at 5 to 15 psi with 22G and 30G nozzles, and multilayer printing on scanned star-shaped targets. In vitro, HDFs, hMSCs, keratinocytes and HUVECs were cultured with Coll, Coll-zMP (zein particles without CaO2), Coll-zOMP and a catalase-containing group under normoxia, hypoxia (~1% O2) or anoxia (< 0.1% O2), and THP-1 monocytes were co-cultured with hMSC-laden bioinks for cytokine arrays and RT-qPCR. In vivo, the bioinks were printed into 6 mm full-thickness wounds in male db/db mice and compared with Tegaderm for 10 days using wound closure, histology, immunostaining, RT-qPCR and spatial transcriptomics.

Key results

The zOMPs had a hydrodynamic diameter of 198.2 ± 10.5 nm. Compared with Coll, Coll-zOMP hydrogels showed lower void fraction, a higher DMT modulus by AFM nanoindentation without a significant change in average surface roughness, and cured height that remained nearly constant when the UV source was moved from 0 to 8 cm. The printed Coll-zOMP hydrogel released oxygen with an initial burst of about 90 μmol/L over the first 23 minutes, followed by about 20 μmol/L at Day 1 and about 5 μmol/L at Day 3 (about 1.98% O2), and H2O2 stayed in a low, non-cytotoxic range over 7 days.

Both bioinks were shear-thinning and kept stable viscosity from 0 °C to about 30 °C. Coll-zOMP formed longer hanging filaments than Coll at all pressures from 5 to 15 psi and kept a filament length of about 40 mm with both 22G and 30G nozzles. INSIGHT printed circles, polygons, spider webs and a seven-layer spiral, and in repeated scan-and-print cycles on a star-shaped target each cycle added about 1 mm of height.

Printed hMSCs remained highly viable after 24 hours at < 0.1% O2. Under oxygen-deficient conditions, HDF migration at 12 hours reached about 60% with Coll-zOMP compared with about 20% with Coll, and keratinocyte colonies were larger and more numerous than on Coll or Coll-zMP. In HUVEC migration, catalase did not remove the benefit of oxygen-generating particles, which the authors took to indicate that oxygen supply, rather than H2O2, is the main contributor. In THP-1 co-culture, Coll-zOMP-hMSCs lowered IL-6 secretion and raised IL10 expression compared with Coll-zMP-hMSCs.

In db/db mice, Coll-zOMP-hMSC wounds closed significantly faster than Tegaderm wounds at Days 3 and 10, with a smaller migrating hyperproliferative epidermis area, fewer neutrophils, fewer CD11b+TNF-α+ cells, more CD11b+CD206+ cells and higher Fn1, Vegfa and Arg1 expression. The HIF-1α-positive area rose from about 6.0% with Tegaderm to 9.9%, 11.3% and 15.7% with Coll, Coll-zOMP and Coll-zOMP-hMSCs. In spatial transcriptomics, migrating epidermis made up 62.5% of spots with Coll-zOMP-hMSC versus 32.0% with Coll, 34 rather than 21 signaling pathways were significant, and Il4, Egf and Grn signaling were specifically enriched alongside an M1-to-M2 pseudotime trajectory.

Open questions

The authors note that interfacial contact was defined by geometry-conforming placement rather than measured tissue adhesion, and they propose lap shear or peel tests. They state that lineage tracing or targeted macrophage depletion is needed to establish causal, cell-intrinsic macrophage reprogramming, and that clinical translation requires long-term bioink function, large-scale manufacturing and storage stability, large-animal studies and evaluation of regulatory and safety issues for AI-assisted bioprinting.

How it connects to other research

An earlier in situ printed scaffold used living microalgae to produce oxygen under light, whereas this bioink generates oxygen chemically from zein-coated CaO2 and adds hMSC paracrine signaling, with a robot that recognizes the wound before printing.