The review considers collagen and gelatin, hyaluronic acid, silk fibroin, chitosan, alginate, fibrin and related matrices. That perspective shifts attention from a scaffold’s nominal composition alone to the physical and biological properties produced by crosslinking. It also supports treating crosslinking as a multidimensional regulator rather than a single measure of scaffold stability.

Different crosslinking methods used in these materials include covalent, photoinduced, enzymatic, ionic, physical and dynamic covalent approaches. The review organizes these approaches around the distinct biological microenvironments they can generate. This comparison matters because the choice of network chemistry is linked to several outcomes at once, including how a material behaves mechanically, how molecules move through it and how its structure changes over time.

A material therefore cannot be characterized adequately through one property when several coupled characteristics may influence its biological performance. Crosslinking is also closely connected to how a scaffold is fabricated. The review examines extrusion bioprinting, electrospinning, freeze-drying, injectable hydrogel formation and porous-scaffold fabrication, emphasizing the coupling between processing routes and network formation.

It further argues that the timing of network creation can be as important as the nominal polymer composition. The central analytical point is that a material’s processing history must be considered when relating its chemistry to its eventual biological environment. Preclinical studies summarized in the review indicate that regenerative performance cannot be inferred from stiffness alone.

Stress relaxation, degradation, transport, interfacial chemistry, immune modulation and preservation of matrix-remodeling capacity are identified as additional considerations. The cited findings are preclinical and therefore do not establish clinical benefit or demonstrate outcomes in patients. The review proposes a mechanistically informed evidence chain to connect the design of a material with its observed behavior.

The sequence begins with material definition, proceeds to properties measured in the hydrated state and then to cellular responses. It extends from those responses to ex vivo or in vivo behavior and, finally, to tissue-specific regenerative outcomes. Reproducibility is another major focus of the proposed validation approach.

The review highlights sterilization, storage and aging-related property drift as factors that may affect a material after fabrication. It calls for controls capable of distinguishing genuine network effects from formulation confounders. Such controls are essential when comparing materials, because an observed difference cannot be assigned to network design if other changes in processing or handling are not adequately addressed.

The review’s final conclusion is that crosslinking should be designed and reported as a multidimensional regulator of the regenerative microenvironment. Its effects on network mechanics, viscoelastic behavior, swelling, transport, degradation, ligand accessibility and cell interactions should be evaluated together with the processing route used to create the material. For regenerative biomaterials, the review argues, crosslinking is not merely a manufacturing operation or a shorthand for stability; it is a central variable linking material chemistry, fabrication and preclinical biological validation.

Subject of Research: Crosslinking, processing and preclinical validation of biopolymer scaffolds for tissue engineering and regenerative medicine

Article Title: Crosslinking-controlled biopolymer biomaterials for tissue engineering and regenerative medicine: network chemistry, processing, and mechanistically informed validation

Article References: Bellucci, S. (2026). Crosslinking-controlled biopolymer biomaterials for tissue engineering and regenerative medicine: network chemistry, processing, and mechanistically informed validation. Frontiers in Bioengineering and Biotechnology, 14, Article 1962375. https://doi.org/10.3389/fbioe.2026.1962375

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1962375

Keywords: crosslinking, biopolymer scaffolds, tissue engineering, regenerative medicine, hydrogels, biofabrication, mechanobiology, matrix remodeling, biomaterials, network chemistry, Crosslinking-controlled, biopolymer

The researchers tested whether the scaffold’s geometry could tune mechanical performance without changing the hydrogel formulation or photocrosslinking conditions. Using melt electrowriting, they produced PLGA fiber lattices with a porosity above 85%, then compared square grids spaced 1,000 and 500 micrometers apart and a 500-micrometer diamond lattice. The finer square grid produced more continuous load-transfer pathways and the highest maximum compressive force during 10 loading cycles. In tensile dynamic mechanical analysis, the square lattice reached a storage modulus above 100 megapascals, approximately one order of magnitude above the diamond design. By comparison, pure GelMA had a storage modulus of about 6 kilopascals. Notably, the PLGA framework represented only 1% by weight of the entire composite, yet it increased stiffness by several orders of magnitude relative to the unfilled gel.

Bioactive glass added a second, biologically relevant form of reinforcement. Particles measuring 20–50 micrometers were mixed into the GelMA precursor at 1, 5, 10 or 20% by weight, and scanning electron microscopy showed that they remained distributed throughout the hydrogel without obvious aggregation. The 1% formulation produced the most favorable balance. It had the lowest swelling, retained its shape after immersion in phosphate-buffered saline and reduced weight loss after 28 days from about 60% for the glass-free composite to approximately 25%. Dynamic mechanical analysis likewise showed an increase in storage modulus at 1% glass, followed by weakening at higher loading, which the authors attributed to particles disrupting continuity in the crosslinked GelMA network. The composite also released calcium, silicon and phosphorus over 21 days as the glass dissolved and the surrounding gel degraded.

Biocompatibility tests showed that the optimum was not simply the formulation with the most glass or the stiffest network. Rat bone-marrow mesenchymal stem cells maintained high viability on the fiber-reinforced composites containing 1 or 5% glass, with the 1% material producing the strongest proliferation signal and cell spreading. High glass contents of 10 and 20% suppressed proliferation during longer culture periods. All formulations produced hemolysis rates below the cited 5% threshold. These results identified P/G@1BG, containing 1% bioactive glass, as the formulation that combined mechanical stability, controlled swelling, sustained ion release and favorable cellular responses. Accelerated alkaline testing also showed that PLGA was more hydrolytically susceptible than polycaprolactone, supporting its selection as a reinforcement intended to lose structural contribution over time.

The optimized composite also accelerated osteogenic differentiation in rat bone-marrow stromal cells. After seven days, alkaline phosphatase activity in cells exposed to P/G@1BG was approximately 1.6 times that of the blank control, and by day 21 the material produced the greatest calcium-mineralization signal in Alizarin Red assays. Molecular measurements showed significant increases in early and late bone-associated genes, including alkaline phosphatase, BMP-2, Runx2, osteopontin, osteocalcin and collagen type I alpha 1. Immunofluorescence at day 14 placed Runx2 mainly in cell nuclei and revealed extensive osteocalcin expression in the cytoplasm. The authors linked these responses to the combined effects of a cell-adhesive GelMA matrix, sustained glass-derived ions and the PLGA scaffold’s guidance of cell organization. The evidence supports enhanced osteogenic differentiation in vitro; it does not by itself establish clinical effectiveness in people.

Pro-angiogenic effects were observed through a second route. P/G@1BG raised the expression of vascular endothelial growth factor A and hypoxia-inducible factor 1-alpha in the stem cells. When the culture medium from treated cells was transferred to human umbilical vein endothelial cells, the P/G@1BG-conditioned medium produced approximately 75.6% closure of a scratch after 24 hours, compared with 45.4% for medium from cells on the glass-free composite and 50.0% for the control medium. The same conditioned medium generated the most extensive capillary-like networks in a six-hour tube-formation assay, with more branching junctions and greater total tube length. These assays indicate that the hydrogel stimulated rat stem cells to release soluble signals that promoted endothelial migration and network formation in vitro.

The researchers next implanted the optimized material into bilateral, full-thickness, 5-millimeter calvarial defects in 18 adult Sprague-Dawley rats, assigning the two defects in each animal as paired sites for a blank control, the glass-free P/G scaffold or P/G@1BG. Specimens were examined at eight and 12 weeks by micro-computed tomography, histology and immunostaining. At 12 weeks, bone coverage reached approximately 60% in defects treated with P/G@1BG, compared with 40% for P/G and 25% for controls. Micro-CT analysis found a bone-volume-to-total-volume ratio of 59.85 ± 7.39%, a relative bone mineral density of 8.00 ± 0.40 times control, a trabecular-number increase of 2.87 ± 0.63 times and a trabecular-separation reduction to 0.56 ± 0.07 times control. Histology showed mature, collagen-rich bone closely associated with the implant in the optimized group, whereas controls contained mostly fibrous tissue and limited peripheral bone.

Markers of both tissue-building systems also rose within the treated defects. At 12 weeks, relative Runx2 and CD31 signals in P/G@1BG defects were 3.0 and 2.8 times those in blank controls, respectively. Double labeling showed persistent spatial association between Runx2-positive osteoblasts and CD31-positive vessels at both eight and 12 weeks, indicating coupled osteogenic and angiogenic activity during healing. The scaffolds progressively degraded while host tissue grew into them, and only mild inflammation was observed. Histological examination of major visceral organs at eight weeks showed no obvious pathological lesions, inflammatory infiltration or necrosis attributable to the material. These findings demonstrate enhanced bone repair and vascularization in a rat cranial-defect model, not yet in human patients or in defects comparable to every form of craniofacial trauma.

RNA sequencing offered a possible molecular explanation for the dual response. Compared with control cells, rat stem cells cultured on P/G@1BG had 784 significantly upregulated and 401 downregulated genes. Enrichment analysis associated the upregulated genes with cell proliferation, extracellular-matrix deposition and signaling pathways including Ras/MAPK. The pathway was also connected to the measured increases in Runx2, osteocalcin, HIF-1-alpha and VEGFA, suggesting that it may participate in osteogenic commitment and the paracrine activation of endothelial cells. The authors were careful not to treat enrichment as proof of causation: additional scaffold controls and experiments that directly disrupt Ras/MAPK signaling would be needed to establish causality. Taken together, the results show how a 3D-printed polymer skeleton, a soft bioactive matrix and glass-derived ions can be allocated different roles, producing a bioresorbable material that supports vascularized bone formation in preclinical testing.

Subject of Research: A mechanically reinforced bioactive hydrogel for osteogenic and angiogenic coupling in craniofacial bone repair

Article Title: Synergistically reinforced bioresorbable hydrogel for mechanical–biological decoupling and osteogenic–angiogenic coupling

Article References: Hong, Y., Lu, H., Luo, J., Li, Y., Ye, Y., Hua, Y., Xu, H., & Xu, T. (2026). Synergistically reinforced bioresorbable hydrogel for mechanical–biological decoupling and osteogenic–angiogenic coupling. Materials Today Bio, 41, Article 103736. https://doi.org/10.1016/j.mtbio.2026.103736

Image Credits: AI Generated

DOI: 10.1016/j.mtbio.2026.103736

Keywords: bioresorbable hydrogel, craniofacial bone repair, melt electrowriting, bioactive glass, PLGA scaffold, osteogenesis, angiogenesis, tissue engineering, rat calvarial defects, Synergistically, reinforced, bioresorbable

Peripheral nerves normally carry communications between the brain and the limbs, but a severe injury can completely interrupt those pathways. Without direct structural guidance, regenerating nerve fibers may lose their direction, leaving downstream muscles without electrical stimulation and vulnerable to permanent atrophy. Surgeons commonly bridge a damaged nerve segment with an autograft, meaning a healthy sensory nerve taken from elsewhere in the same patient.

Although this approach is clinically established, it requires sacrificing another nerve and can leave donor-site numbness, chronic pain or a reduced supply of usable tissue. Hollow plastic conduits avoid that surgical cost, but the release notes explain that wider gaps can be difficult to cross because the tubes may not reproduce the physical cues and biochemical support needed by growing axons. The team addressed that problem by integrating three fabrication techniques on one direct-writing platform: melt writing for the structural outer layer, electrospinning for the porous middle membrane and low-temperature cell patterning for the innermost living tracks.

The conduit was built continuously rather than transferring fragile components between separate machines. Its outer layer consists of an open polycaprolactone mesh that functions as an exoskeleton and resisted compression and suturing forces of up to 1.64 N. The middle layer is a nanofiber membrane with pores averaging 0.55 micrometers across. These openings permit the movement of oxygen and nutrients while discouraging scar-forming connective tissue from entering the conduit.

The innermost layer contains Schwann-cell-bearing microfilaments, creating a biological trackway for nerve regrowth. At the core of the work was a bioprinting challenge involving the printing of living Schwann cells, the supporting cells that normally help insulate and direct peripheral nerve fibers. Electro-assisted bioprinting can pull cell-containing ink through fine nozzles into microfibers, but high-resolution printing creates a damaging trade-off.

Viscous inks tend to retain their deposited shape, yet forcing them through narrow nozzles produces strong shear stress that can rupture cell membranes. Diluting the ink reduces that stress, although the resulting filaments can collapse into puddles after deposition. Chemical hardeners may be toxic to the cells, while the relatively high voltages used to form fine fibers can also damage living material.

The researchers managed this conflict with temperature. Their bioink contained alginate, polyethylene oxide and thermosensitive gelatin methacryloyl, together with living Schwann cells. The nozzle was held at 25 °C, where the mixture was fluid enough to pass through the nozzle without imposing the shear associated with a thicker formulation.

A chilled collection plate at 4 °C rapidly stabilized each deposited jet before it could spread. Under a comparatively gentle electric field of 1.0 kilovolt, the system produced continuous microfilaments measuring about 36 micrometers in diameter. Temperature therefore served as a way to control the material’s flow during printing and its stability immediately after deposition.

In laboratory cultures, nerve cells attached to the printed tracks organized into dense, directionally aligned ribbons rather than dispersing randomly. RNA sequencing suggested a molecular explanation for this behavior. Contact with the microfilaments, together with chemical signals supplied by the Schwann cells, activated axon-guidance genes including Ephb1 and Gnai1.

Those signals were associated with cytoskeletal programs that steer elongating axons. At the same time, expression linked to programmed cell death and routine cell proliferation decreased, while the cells redirected their activity toward extending elongated signaling structures. These observations concern laboratory cultures and gene-expression patterns; the release does not report a separate clinical assessment of the underlying molecular mechanisms.

When implanted across 10 mm gaps in rat sciatic nerves, the composite conduits were evaluated over 16 weeks against natural nerve autografts and conduits without living cells. Regenerating nerve bundles in the living devices developed myelin insulation averaging 1.02 micrometers in thickness, close to the 1.15-micrometer average measured in the autografts. Repaired nerves in the living-conduit group conducted impulses at 5.67 meters per second, compared with 5.88 meters per second for the autograft control.

The living-conduit rate was nearly double the rate measured in the acellular conduits. These findings indicate comparable functional performance between the two repair strategies in this rat model, rather than proof of equivalence in people. The experiment also shows that the living patterned tracks supported a different outcome from empty conduits, although the release does not provide a numerical conduction rate for the acellular group.

Functional recovery extended beyond the measured conduction velocity. Severed connections normally leave distal calf muscles vulnerable to denervation atrophy. The living conduits preserved approximately 49% of distal calf muscle wet weight, whereas empty conduits preserved less than 24%.

Walking trials also showed restoration of normal hindpaw coordination in animals receiving the living conduits. Together, the electrophysiological and behavioral results suggest that the engineered bridge did more than provide a passive path: the patterned Schwann-cell tracks supported organized nerve regrowth strong enough to reduce downstream muscle loss and recover coordinated movement during the study period. The reported results are therefore not limited to an electrical measurement; they include anatomical evidence of myelinated bundles and functional measures of muscle preservation and movement.

The study offers a manufacturing proof of concept for multilayered implants containing living cells, but the evidence remains preclinical. Before human testing, the researchers will need to test the conduits across gaps measured in multiple centimeters in large animal models. The team must also establish low-temperature sterilization methods that do not kill the embedded Schwann cells and adjust the degradation rate of the polycaprolactone shell so that it disappears at a pace suited to the slower timetable of human nerve healing.

The reported results support further large-animal validation, not an immediate clinical recommendation. If that next phase succeeds, the approach could offer an alternative for patients who cannot donate another healthy nerve or do not have enough tissue for a conventional autograft. For patients, the significance of the work is that it combines several functions in one device: mechanical support, selective exchange with the surrounding environment and a living, aligned substrate for nerve fibers.

The conduit is not simply a hollow tube, because its inner tracks are populated with cells that can provide biochemical cues while the printed architecture supplies physical direction. At the same time, the outer polycaprolactone layer is designed to withstand handling while the middle membrane limits inward connective-tissue growth. The result is a compact example of how melt writing, electrospinning and low-temperature bioprinting can be combined without moving delicate constructs between separate machines.

The study’s central claim is a preclinical one, supported in rats: under the reported conditions, a living bioprinted conduit supported nerve regeneration over a 10 mm gap with functional outcomes close to those of a natural autograft. Its broader usefulness will depend on validation across longer gaps, sterilization methods compatible with living cells, shell degradation matched to healing time, and eventual testing in humans.

Subject of Research: Preclinical bioprinted nerve conduits for peripheral nerve regeneration in rats

Article Title: Living "railway tracks" bioprinted inside artificial conduits match gold-standard nerve repair

Article References: Living "railway tracks" bioprinted inside artificial conduits match gold-standard nerve repair. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: peripheral nerve repair, 3D bioprinting, Schwann cells, nerve guidance, tissue engineering, sciatic nerve, rat study, aligned microfilaments, biofabrication, electrospinning, Living, railway

The results indicate that the cellular origin of the matrix can determine how perlecan-associated sugars influence stem-cell state and multilineage differentiation. The study focused on adult infrapatellar fat pad-derived stem cells, or IPFSCs, isolated from six de-identified young donors and pooled for the experiments. These cells have an inherent propensity for cartilage formation and can deposit extracellular matrix rich in components of the basement membrane.

The researchers generated two types of cell-derived extracellular matrix: dE from unmodified IPFSCs and s-dE from cells immortalized with the SV40 large T antigen. The dECM produced by the immortalized cells had previously been found to contain higher perlecan expression and to be more effective at supporting chondrogenic differentiation than dECM from unmodified cells. This comparison provided the basis for asking whether the functional contribution of perlecan’s sugar chains was also dependent on the cellular source of the matrix.

Perlecan is a multidomain proteoglycan that forms part of basement membranes and other pericellular matrices. Its N-terminal domain carries heparan sulfate glycosaminoglycan chains, which can store and present growth factors such as FGF-2 and BMP-2. The study used heparinase III, or HPA, to remove these chains selectively from matrix-resident perlecan while leaving the protein’s core intact.

HPA was applied to dECM substrates at 0.04 U/cm² for one hour at 37°C, while control substrates received buffer under the same conditions. Immunofluorescence supported the treatment: signal from the antibody 10E4, which recognizes intact heparan sulfate, declined markedly in treated matrices, while signal from 3G10, which recognizes the stub left after enzymatic cleavage, appeared. Strong perinuclear 10E4 staining remained in the cells, consistent with the enzyme reaching extracellular matrix but not newly synthesized intracellular chains.

Five experimental groups were compared: standard tissue-culture plastic, unmodified-cell matrix, heparinase-treated unmodified-cell matrix, immortalized-cell matrix and heparinase-treated immortalized-cell matrix. The cells were preconditioned on these surfaces for about 10 days before stemness markers and differentiation responses were measured. Most assays were conducted with at least three technical replicates, although the cells were pooled from six donors, so the experiment did not provide six independent donor-level estimates.

Gene expression was assessed by quantitative reverse-transcription PCR, surface markers by flow cytometry, and cartilage, fat and bone differentiation through 21-day induction protocols. Chondrogenic induction used three-dimensional pellets under 5% oxygen, adipogenic and osteogenic assays were conducted on plastic, and the resulting phenotypes were measured with molecular, histological and colorimetric readouts. Untreated dE and s-dE altered the expression of several stemness-associated genes relative to plastic.

Both matrices increased SOX2, NANOG and NES, with larger increases generally seen on s-dE, while MYC, NOV, BMI1 and KLF4 were lower. Heparinase treatment attenuated SOX2, NANOG and NES in both matrix conditions and increased NOV. Some responses were source-specific: MYC rose relative to dE after its chains were removed but fell relative to s-dE in the corresponding comparison, and KLF4 was selectively reduced in the treated immortalized-cell matrix condition. Flow cytometry also produced divergent marker profiles.

Heparinase treatment lowered SSEA4 positivity in the unmodified-cell model, but raised CD90, CD146 and SSEA4 positivity in the immortalized-cell model, which had the highest CD146 and SSEA4 percentages among all groups. CD73 positivity remained near 100% across the conditions. Both untreated dE and s-dE suppressed fat formation relative to plastic, producing lower expression of LPL, FABP4, CEBPA and PPARG and less lipid accumulation by Oil Red O staining.

The measured Oil Red O absorbance was 0.214 ± 0.005 for dE, 0.229 ± 0.004 for s-dE and 0.431 ± 0.008 for plastic. The treated groups accumulated more lipid droplets, with absorbance values of 0.342 ± 0.016 for dE(H) and 0.395 ± 0.007 for s-dE(H), and showed increased expression of all four adipogenic markers. Because this response occurred on both matrices, it appears less dependent on the source of the matrix than the cartilage response was.

The authors suggest that heparan sulfate chains may contribute to the anti-adipogenic influence of perlecan through their ability to sequester growth factors, although the experiment did not directly measure which signals accounted for the effect. Chondrogenic responses were strongly dependent on both matrix source and chain removal. After 21 days in three-dimensional pellet culture, cells preconditioned on untreated dE or s-dE expressed more SOX9, ACAN and COL2A1 than cells expanded on plastic and produced stronger Alcian blue and Safranin O staining, consistent with greater accumulation of sulfated glycosaminoglycans.

The untreated matrices also yielded larger pellets. Removing heparan sulfate from dE reduced SOX9, ACAN and COL2A1 relative to untreated dE and diminished the histological staining. In contrast, s-dE(H) produced the largest pellets and most prominent sulfated-glycosaminoglycan deposition of the matrix conditions.

Its day-21 expression values for SOX9, ACAN and COL2A1 were markedly higher than those of cells preconditioned on untreated s-dE: 11.713 ± 0.164, 14.310 ± 0.891 and 111,695.573 ± 5,567.585, respectively. The corresponding dE(H) values were 6.458 ± 0.075, 6.009 ± 0.166 and 38,001.631 ± 1,339.186. The osteogenic response was even more selective.

Alizarin Red S staining detected mineralized matrix exclusively in cells preconditioned on s-dE(H) after 21 days of induction, with an absorbance of 0.766 ± 0.029, whereas the other groups showed minimal mineralization. The molecular results did not follow a simple pattern: RUNX2, SP7, BGLAP and ALPL changed over time, and s-dE(H) did not show a corresponding increase in BGLAP despite its strong mineral deposition. The authors interpret this as a possible temporal separation between gene-expression peaks and matrix formation.

They also note that elevated CD146 and SSEA4 in s-dE(H) could be relevant because these markers have been associated with differentiation-competent progenitor populations, but the experiment did not establish that marker-positive cells caused the mineralization. The results therefore support a selective association between the treated immortalized-cell matrix and osteogenic output, while leaving its cellular mechanism unresolved. The authors propose that heparan sulfate chains from normal and immortalized-cell matrices differ in structure and signaling activity.

One possibility is that chains on unmodified-cell perlecan store FGF-2 in a way that supports a self-renewing state and cartilage formation, so their removal weakens those signals. Chains in s-dE may instead restrict receptor activation, with their cleavage revealing or enabling lineage-promoting interactions. BMP-2 binding, Wnt-related effects and changes in matrix mechanics could also contribute.

These remain hypotheses because the investigators did not directly characterize chain length, sulfation patterns, growth-factor release or receptor activation, and they did not block FGF or BMP signaling to establish causality. They also did not determine whether heparinase altered the stiffness or elasticity of the two matrices, meaning that biomechanical differences cannot be separated from biochemical effects in the observed differentiation outcomes. Taken together, the findings identify matrix-resident perlecan heparan sulfate as a context-dependent regulator of IPFSC behavior rather than a uniform brake or accelerator of differentiation.

Its removal consistently favored adipogenesis, but its effect on cartilage formation depended on whether the matrix came from unmodified or SV40LT-immortalized cells, and osteogenic mineralization occurred only after preconditioning on the treated immortalized-cell matrix. The findings provide a rationale for engineering decellularized matrices with defined glycosaminoglycan architecture, but they also show why the matrix must be evaluated as a complete biochemical and mechanical environment. Direct glycomic analysis, measurements of growth-factor availability, pathway-specific inhibition and controlled comparisons of substrate stiffness are needed before the molecular mechanism or translational potential can be established.

Because the work was performed entirely in vitro with pooled donor-derived cells, the results do not yet establish safety, efficacy or clinical benefit in people or in an animal model of tissue repair.

Subject of Research: Source-dependent effects of perlecan heparan sulfate on human stem-cell differentiation in decellularized extracellular matrix

Article Title: Source-dependent perlecan heparan sulfate chains in dECM modulate stem cell fate and multilineage differentiation

Article References: Wei, Z., Yu, Q., Shahriar, A., Xie, X., Chen, S., & Pei, M. (2026). Source-dependent perlecan heparan sulfate chains in dECM modulate stem cell fate and multilineage differentiation. Frontiers in Bioengineering and Biotechnology, 14, Article 1930107. https://doi.org/10.3389/fbioe.2026.1930107

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1930107

Keywords: stem cells, decellularized extracellular matrix, perlecan, heparan sulfate, chondrogenesis, osteogenesis, adipogenesis, infrapatellar fat pad, heparinase III, tissue engineering, Source-dependent, heparan

The research team, led by members of professor Eben Alsberg’s laboratory in UIC’s College of Engineering, set out to solve one of the most stubborn problems in tissue engineering. Human tissues are not flat. Cartilage curves around joints, blood vessels branch and bend, the trachea spirals in a series of horseshoe-shaped rings, and bone sweeps through the skeleton in arcs that no simple mold can reproduce. Conventional biofabrication approaches, which typically build tissues layer by layer or cast them in static scaffolds, have struggled to reproduce these intricate curvatures and fine surface features. The new strategy instead lets the material do the work, programming a flat construct to bend, fold, and curl itself into a target geometry as its internal structure swells at different rates.

Kirigami, the Japanese art of paper cutting, has already inspired engineers far beyond the classroom. Its techniques have informed deployable structures for spacecraft, flexible semiconductors, wearable technology, and optical devices. The appeal is straightforward: by introducing carefully placed cuts into a flat, stiff sheet, the material gains stretchability and flexibility it never had before, and it can be reshaped without any change to its chemical composition. What had been largely unexplored, according to Kaelyn L. Gasvoda, the paper’s lead author and a 2024 UIC PhD graduate now at the Mayo Clinic, was whether kirigami could be adapted to tissue engineering and, crucially, to soft, gel-like materials that can host living cells.

The challenge is that living hydrogels are nothing like paper. They are fragile, they swell in aqueous culture media, and any structural trick must remain cytocompatible, meaning it cannot harm the cells embedded within it. The UIC team’s solution was a hydrogel platform built from derivatives of gelatin and alginate, a natural polymer derived from seaweed. Both materials are well established in the biomaterials field: gelatin offers cell-friendly biochemical cues, while alginate provides a tunable structural backbone. By combining them, the researchers created a material system in which mechanical behavior and biological support could be engineered separately and then brought together.

The key to programming shape change lies in a technique called photolithography. Using patterned light, the team created regions of different crosslinking densities within the same hydrogel construct. Crosslinks are the molecular bridges that tie polymer chains together, and their density dictates how much a given region can swell when immersed in culture medium. Densely crosslinked areas resist expansion and hold their form, while lightly crosslinked areas absorb the medium and grow. By patterning these regions with kirigami-inspired precision, the researchers could dictate exactly which parts of a construct would stay rigid and which would expand, and by how much, producing controlled bending, folding, and self-patterning over time.

The design philosophy behind the platform reflects a deliberate division of labor within the material. Ding, a research associate professor on the team, explained that the researchers aimed to create structures with a rigid outer framework that would maintain its shape without expanding in the culture medium. In contrast, the inner regions needed to absorb the medium and expand while remaining compatible with long-term cell survival, growth, and differentiation. That combination, a stable skeleton paired with a growing, living interior, is what allows the constructs to transform autonomously while the cells inside them continue to develop into tissue-like structures with precisely defined geometries, all without additional manipulation by the researchers.

Gasvoda described the progression as an effort to push beyond the team’s earlier demonstrations. The initial achievement of constructs that changed shape over time was exciting, she said, but the goal was to create more complex shapes with controlled self-patterning and bending, designing structures where the researchers could precisely guide how they form and transform. That precision matters because, as Ding emphasized, complex geometries are central to tissue engineering: human tissues have intricate three-dimensional shapes that are closely linked to their functions. The Alsberg laboratory focuses on engineering tissues with complex architectures, including cartilage, bone, tracheal, and vascular tissues, with the aim of recreating their native structures and functions rather than approximations of them.

The implications extend to wound healing as well as tissue replacement. Repairing defects in curved anatomical surfaces, whether in cartilage, skin-adjacent structures, or tubular organs, requires implants or engineered constructs that match the geometry of the site they are meant to restore. A flat patch forced onto a curved wound bed fits poorly, integrates poorly, and functions poorly. A construct that can be fabricated flat, shipped and handled easily, and then programmed to assume the correct curvature as it matures offers a fundamentally different approach. The platform also enables the spatial organization of multiple cell types within a single construct, supporting their distinct functions in ways that uniform scaffolds cannot.

What makes the work particularly striking is its conceptual economy. No new chemistry was required, no exotic materials, and no external actuators. The shape change emerges from the interplay between patterned crosslinking, differential swelling, and time, the same interplay that turns a folded sheet of paper into a snowflake. Gasvoda summarized the demonstration succinctly: kirigami-patterned, cytocompatible hydrogels can be engineered to autonomously bend, fold, and transform into complex three-dimensional shapes. In doing so, the approach addresses a longstanding challenge in the field, opening the possibility of engineering tissues that more closely replicate the intricate architectures of the native tissues they are meant to replace or repair.

The team is candid about the road ahead. Having shown that the platform can generate complex geometries in concert with new tissue formation in the laboratory, the next step is to evaluate its performance in animal models, with the long-term goal of translating the technology toward clinical applications. That path will test whether the programmed transformations remain reliable in the messier environment of a living body, where mechanical loading, biochemical signaling, and immune responses all compete with the engineered design. But the underlying idea, that a flat sheet of living material can be taught to fold itself into the shape of the tissue it will become, offers a compelling glimpse of where biofabrication may be headed, and it began, fittingly, with a pair of scissors and a sheet of paper.

Subject of Research: A 4D bio-kirigami hydrogel platform for engineering complex tissue curvatures

Article Title: New 4D biofabrication technology holds promise for tissue engineering, wound healing

Article References: New 4D biofabrication technology holds promise for tissue engineering, wound healing. Original publication (Bibliographic details unavailable.)

Image Credits: AI Generated

DOI: Not provided

Keywords: 4D biofabrication, kirigami, hydrogels, tissue engineering, wound healing, photolithography, alginate, gelatin, cell Biomaterials, University of Illinois Chicago, cartilage, regenerative medicine

A team of researchers from Aalborg University and Aalborg University Hospital in Denmark, working with collaborators in Qatar and the United States, has now reported the first controlled in vivo evaluation of a trilayer composite electrospun scaffold for urethral reconstruction. The construct, designated CPC, consists of a core of poly(L-lactide-co-glycolide), or PLGA, sandwiched between two layers of chitosan, a naturally derived polysaccharide. The design rationale is biomimetic: the luminal chitosan layer is intended to mimic the urethral mucosa, providing a hydrophilic, bioactive surface that supports epithelial cell attachment and migration while contributing the antimicrobial properties characteristic of chitosan-based biomaterials. The central electrospun PLGA layer serves as the mechanical backbone, analogous to the submucosal tissue of the native urethral wall, offering tunable tensile strength and gradual hydrolytic degradation over the healing timeframe. The outer chitosan layer interfaces with the surrounding corpus spongiosum, the vascular tissue that envelops the urethra, promoting cellular infiltration and angiogenesis.

The biological logic behind the chitosan layers rests on well-documented immunomodulatory chemistry. Chitosan oligosaccharides act on macrophage pattern-recognition receptors to drive polarization of these immune cells toward the M2 phenotype, the anti-inflammatory state associated with tissue repair rather than with destruction. M2 macrophages secrete pro-angiogenic factors including vascular endothelial growth factor, or VEGF, supporting early vascularization of healing tissue. Chitosan additionally confers hydrophilicity and antimicrobial activity that complement the structural advantages of synthetic polyester cores. The hypothesis was that this layered architecture would attenuate the foreign-body response and support vascularization while preserving the mechanical integrity needed for surgical handling. Prior in vitro work on a related trilayer chitosan/poly(L-lactide)/chitosan construct had demonstrated superior mechanical strength, suture retention, smooth muscle cell growth, and angiogenic potential compared with monolayer PLA scaffolds, motivating the present in vivo evaluation. The Danish team substituted PLGA for PLA in the core layer because PLGA’s degradation rate can be tuned through the lactide-to-glycolide ratio, offering more flexibility than the slower-degrading PLA, and because PLGA carries broad regulatory precedent in absorbable medical devices.

What distinguishes this study from much of the preclinical urethral tissue-engineering literature is its methodological rigor. A 2021 systematic review of 28 rabbit studies published between 2014 and 2020 found a mean adherence of only 53 percent to the ARRIVE guidelines for reporting animal research, with no included study reporting sample-size estimation methodology and the majority lacking blinded outcome assessment. The new study was designed and reported in accordance with ARRIVE 2.0, directly addressing each of these documented gaps. The researchers performed an a priori sample-size calculation, prespecified a continuous quantitative primary endpoint rather than the binary outcomes such as fistula formation that have constrained formal power calculation in earlier work, and implemented blinded outcome assessment throughout. Twenty-four male New Zealand White rabbits were allocated in a one-to-one-to-one ratio to dorsal onlay urethroplasty with the trilayer CPC scaffold, with a monolayer PLGA scaffold, or to a sham surgical control, using weight-stratified allocation. Surgeons were blinded to group allocation during urethral exposure and defect creation, and because both scaffolds were thin, white, semi-flexible electrospun sheets that were not reliably distinguishable on inspection or handling, surgeons remained effectively blinded to scaffold type throughout the procedure. Retrospective misidentification of scaffold type was common when surgeons were informally asked to guess, supporting the integrity of the blinding.

The surgical model itself mirrors adult substitution urethroplasty technique. Following urethral catheterization, the penile urethra was exposed through a ventral midline skin incision, mobilized, and a standardized dorsal urethral defect measuring 10 by 3 millimeters was created. In scaffold-treated groups, a dorsal onlay graft of identical dimensions was placed on the defect and sutured with interrupted 6-0 polydioxanone monofilament sutures. To divert urine and protect the healing site, a perineostomy was created proximal to the urethroplasty, a strategy preferred over indwelling catheterization because rabbits do not reliably tolerate prolonged urethral catheters. The primary endpoint was vessel density on CD31 immunohistochemistry at 12 weeks, with secondary endpoints including tissue thickness, fibrosis, smooth muscle content, re-epithelialization, inflammatory cell scores, and urethral patency assessed by post-mortem retrograde urethrography. All histological assessments were performed by a single pathologist blinded to treatment allocation, and intra-observer reliability of vessel-density scoring was excellent, with an intraclass correlation coefficient of 0.97.

The results told a more nuanced story than the hypothesis predicted. Vessel density differed significantly between treatment groups overall, with sham animals exhibiting the highest density at a median of 78.6 vessels per square millimeter, monolayer PLGA the lowest at 39.1, and the trilayer CPC intermediate at 46.3. The direction of effect matched the in vitro pro-angiogenic activity reported for chitosan-containing constructs, and the observed CPC-PLGA median difference of 7.2 vessels per square millimeter closely matched the prespecified clinically meaningful difference of seven used in the sample-size calculation. However, the within-group variability substantially exceeded the planning assumption, reducing statistical power below the design target and widening the 95 percent confidence interval around the CPC-PLGA difference to span from minus 27.3 to 41.6 vessels per square millimeter. The trilayer did not significantly differ from the monolayer, and the overall between-group variation was driven by higher values in sham animals than in PLGA-treated animals. The authors interpret the sham finding carefully: the rabbit urethra revascularizes robustly after primary closure of a small dorsal defect, so the sham group represents an upper bound for vascularization in this model rather than a vascularization-deficient comparator.

Perhaps the most striking quantitative finding was a coherent host-response signature linking vascularization, tissue thickness, and fibrosis. Vessel density was inversely correlated with tissue thickness and with fibrosis, while tissue thickness was positively correlated with fibrosis. This pattern describes an axis of host response in which scaffold-supported remodeling produced thicker, more fibrotic, and less vascularized tissue, consistent with the behavior of synthetic scaffolds at intermediate timepoints, where a sustained foreign-body response can shift the balance from constructive vascularization toward fibrotic encapsulation. The thickness finding also carries a metric caveat: scaffold-driven regeneration produced thicker tissue at the implantation site over which a comparable absolute vessel population was distributed, biasing the per-area vessel density downward. Fibrosis itself did not differ significantly between groups, with median collagen area near 82 to 84 percent across all arms, a near-equivalence in the absence of any scaffold material suggesting that the dominant contributor to the measured collagen signal was native urethral and adjacent corpus cavernosum collagen captured within the region of interest rather than scaffold-induced scar formation.

On the inflammatory front, scaffold implantation elicited a foreign-body response in both treatment groups, with significantly elevated multinucleated giant-cell scores compared with sham. Yet the macrophage comparison provided the most defensible signal in favor of the trilayer design: median macrophage scores were lower in CPC than in PLGA, a difference that trended toward but did not reach statistical significance. This pattern is consistent with chitosan attenuating the foreign-body response, possibly through its established capacity to favor M2 over M1 macrophage polarization via chitosan-oligosaccharide signaling, while PLGA implants drive a stereotyped macrophage and giant-cell signature partly through acidic by-products of polymer hydrolysis. The authors are careful to note that this chitosan-mediated modulation is inferred from semi-quantitative macrophage counts rather than direct phenotypic markers, and that the elevated giant-cell counts in scaffold-treated groups cannot be classified as uniformly pro-inflammatory without immunohistochemistry for canonical M1 and M2 polarization markers. Meanwhile, re-epithelialization was essentially complete across all groups, with median urothelial coverage of 100 percent in the CPC group, and no animal demonstrated ulceration, unresolved wound bed, or tissue necrosis at the 12-week endpoint. The few focal regions of missing urothelium lacked the edge reaction, granulation tissue, and inflammatory infiltrate characteristic of true ulceration and were interpreted as denudation of mature urothelium, possibly a processing artifact.

Functionally, the news was uniformly good. Retrograde urethrography demonstrated preserved patency in all 23 animals completing the planned 12-week endpoint, with no fistulae or contrast extravasation observed. No animal met the prespecified stricture criterion of a 50 percent or greater reduction in urethral diameter at the implantation site, and lumen-width ratios were numerically wider in scaffold groups than in sham, a directional pattern consistent with the structural role of an onlay graft in providing a template for lumen-supporting tissue regeneration during spongiosal fibrosis. No postoperative complications or infections were observed during follow-up, and no macroscopic fistulae, adhesions, necrosis, or calculi were seen at harvest. One sham animal was euthanized early because of acute-onset bilateral hindlimb paralysis, judged unrelated to the experimental intervention, and no animals met exclusion criteria during acclimatization.

The authors conclude that the trilayer scaffold was feasible to implant and well tolerated, supporting tissue regeneration with preserved urethral patency, but that the findings refine rather than confirm the development hypothesis. They identify several priorities for future work: earlier endpoints at two and four weeks to directly characterize the chitosan-mediated angiogenic and immunomodulatory window, M1/M2 macrophage and giant-cell phenotyping to test the polarization hypothesis, mechanical-modulus tuning to match the elastic modulus of native urethral tissue at approximately 25 kilopascals, and modulation of the chitosan-to-PLGA mass ratio as a further design lever. Crosslinking strategies to slow chitosan resorption could extend the bioactive window of the surface layers, and translational evaluation will ultimately require larger animal models with urethral anatomy closer to humans, longer follow-up, and head-to-head comparison against the autologous buccal mucosa graft that remains the clinical reference standard. The rabbit dorsal onlay model itself demonstrated an excellent safety profile across all groups, supporting its utility as a reproducible platform for controlled comparison of subsequent urethral scaffolds, and the biological criteria validated here, including complete re-epithelialization without ulceration and preserved patency, are of considerable interest for hypospadias-directed scaffold development, where excessive inflammation and fibrosis contribute to fistula formation and repair failure.

Subject of Research: In vivo evaluation of a chitosan/PLGA/chitosan trilayer electrospun scaffold versus monolayer PLGA for urethral reconstruction in rabbits

Article Title: In vivo evaluation of a chitosan/PLGA/chitosan composite versus monolayer PLGA electrospun scaffold for dorsal onlay urethroplasty: a controlled study in rabbits

Article References: Mahdi, M. B., Mohtaram, F., Haase, R. N., Tuckus, G., Fojan, P., Bergman, J., Thomsen, C., Kjærgaard, B., Sørensen, S. R., Alipour, H., Abbas, T. O., Pennisi, C. P., & Nielsen, T. K. (2026). In vivo evaluation of a chitosan/PLGA/chitosan composite versus monolayer PLGA electrospun scaffold for dorsal onlay urethroplasty: a controlled study in rabbits. Frontiers in Bioengineering and Biotechnology, 14, Article 1891449. https://doi.org/10.3389/fbioe.2026.1891449

Image Credits: AI Generated

DOI: 10.3389/fbioe.2026.1891449

Keywords: urethroplasty, urethral stricture, chitosan, PLGA, electrospun scaffold, tissue engineering, vascularization, foreign-body response, rabbit model, regenerative medicine, biomaterials, hypospadias

The review’s central premise is that adhesion is not a passive glue but an active signaling hub. When a cell contacts the extracellular matrix, transmembrane receptors called integrins bind to motifs such as the arginine-glycine-aspartate, or RGD, sequence found in fibronectin and other matrix proteins. This binding event nucleates a focal adhesion, a nanoscale assembly of more than a hundred proteins including talin, vinculin, paxillin, kindlin and focal adhesion kinase. Talin acts as the critical mechanical link, stretching under force to expose binding sites for actin filaments of the cytoskeleton. Vinculin reinforces this linkage, and the review highlights recent work measuring the mechanical response of vinculin directly, showing how the protein unfolds and refolds under tension to tune adhesion strength. The result is a molecular clutch that connects the outside world to the cell’s internal scaffold.

What makes focal adhesions remarkable is their layered architecture. Cited work in the review describes a stratified organization in which integrins at the membrane face, adapter proteins in the intermediate zone and actin regulators at the deepest layer transmit force across tens of nanometers with remarkable fidelity. Single-molecule tension sensors, including DNA-based probes and plasmonic nanosensors, have now revealed that individual adhesion bonds experience piconewton-scale forces that fluctuate over milliseconds. A 2026 study cited by the authors linked molecular tension to cellular traction forces in a multiscale analysis of focal adhesion mechanics, confirming that the forces cells exert on their surroundings are built up from the sum of these molecular-scale events. Computational models of force-dependent adhesion assembly and disassembly further show how adhesions can self-stabilize under load, a generic mechanism that helps cells maintain grip in a mechanically noisy environment.

Downstream of the adhesion complex, mechanical signals cascade into the cytoskeleton and ultimately into the nucleus. The Rho family of small GTPases, acting through effectors such as Rho-associated protein kinase, or ROCK, and the beta-PIX/GIT complex, remodels actin filaments in response to adhesion cues. Focal adhesion kinase activates signaling pathways including JNK, which the review notes is activated by mechanical stress in an integrin-dependent manner. One of the most consequential endpoints is the transcriptional co-activator YAP, the nuclear messenger of the Hippo pathway. Recent work cited in the review demonstrates that FAK, vinculin and talin jointly control mechanosensitive YAP nuclear localization, meaning that the physical state of adhesions directly determines which genes the cell expresses. In endothelial cells, substrate stiffness modulates function through a YAP-Dll4-Notch1 pathway, illustrating how mechanical information flows all the way from the matrix to developmental signaling.

The review devotes substantial attention to substrate stiffness, one of the most influential discoveries in modern cell biology. The landmark finding that matrix elasticity directs stem cell lineage specification, published nearly two decades ago, showed that mesenchymal stem cells become bone-like on stiff substrates and brain-like on soft ones, purely as a function of the elasticity beneath them. The new review extends this picture with recent evidence that substrate stiffness alters the layer architecture and biophysics of human induced pluripotent stem cells, modulating their differentiation potential. It also complicates the story: polyacrylamide hydrogels with rigidity-independent surface chemistry show limited long-term maintenance of pluripotency on soft substrates, suggesting that stiffness sensing and surface chemistry interact in ways that are still being untangled. Cardiomyocytes, fibroblasts and cancer cell lines each respond to stiffness gradients in distinct ways, and the review emphasizes that some stiffness sensing by fibroblasts is myosin-independent, regulated instead by the viscoelasticity of flowing actin networks.

Beyond stiffness, the authors catalog the experimental toolbox that mechanobiologists now use to interrogate adhesion. Micropatterning techniques constrain cell shape and ligand spacing on engineered surfaces, and studies cited in the review show that cell shape and tension alone can alter focal adhesion structure, while biomimetic micropatterned surfaces change the adhesion and morphology of cervical cancer cells. Titanium dioxide nano-biopatterning has revealed optimal ligand presentation for cell-matrix adhesion formation. Stretch devices apply cyclic strain to cells grown on flexible membranes, and dimensionality matters: mechanical stretch regulates cell behavior differently in two-dimensional culture than in three-dimensional hydrogels. Recent work on cell-matrix feedback shows that stretch-induced cellular memory and fibroblast activation depend on the mechanical properties of the matrix itself, hinting that cells remember the forces they have experienced through persistent changes in their adhesion machinery.

Chemical strategies complete the toolkit. Thermoresponsive polymers allow reversible cell adhesion that can be switched on and off by temperature, a property exploited in cell sheet engineering. Reversible host-guest interactions in three-dimensional microscaffolds permit mechanical stimulation of single cells with exquisite spatial control, and photo-switchable surfaces enable spatiotemporal modulation of cytoskeletal forces that visibly deform the nucleus. Amphiphilic cationic cyclodextrin nanovesicles serve as versatile cues for guiding cell adhesion, and strain-dependent glutathionylation of fibronectin fibers has been shown to prime an integrin switch, a striking example of chemistry and mechanics coupling within the matrix itself. Even the extracellular domains of E-cadherin, the prototypical cell-to-cell adhesion molecule, determine mechanical phenotypes of epithelia through outside-in signaling, underscoring that the review’s framework spans both cell-matrix and cell-cell junctions, where cadherins and F-actin engage in a positive feedback loop initiated by cortical tension.

Measurement technology occupies a special place in the review, particularly bio-atomic force microscopy. AFM can map the local stiffness of living cells, probe adhesion forces with piconewton sensitivity and, in newer modes such as nanoendoscopy, visualize intracellular nanostructures in living cells. Fluidic force microscopy allows simultaneous measurement of single-cell mechanics and adhesion to materials, while atomic force acoustic microscopy extends mechanobiological analysis to manifold live cell types in vitro. The integration of AFM with fluorescence microscopy, including FRET-based tension probes and super-resolution imaging of DNA-based tension sensors, is transforming the field’s ability to watch mechanotransduction happen in real time. Deep learning-assisted AFM image recognition now enables efficient single-cell mechanics measurements even in co-culture environments, and roughness-driven compressive sensing improves nanoscale surface characterization. The review positions these quantitative methodologies as the connective tissue of adhesion mechanobiology, the tools that turn qualitative observations into testable, predictive models.

The applications flowing from this unified framework are broad. In tissue engineering, scaffold-free three-dimensional-printed heart tissue grafts develop better under mechanical stimulation, and micropatterned nanofiber scaffolds guide muscle tissue formation. Biomechanical stimulation of mesenchymal stem cells in three-dimensional peptide nanofibers promotes bone differentiation. In cancer biology, adhesion mechanics govern how tumor cells migrate through constrained microenvironments, and engineered T cells are being designed to navigate the structurally complex terrain of tumors. Adhesion signaling is also entwined with cellular senescence, with integrin-mediated adhesions regulating the entry of cells into aged, dysfunctional states, a connection with implications for age-related diseases such as emphysema. By integrating mechanical stimulation strategies, chemotransduction, adhesion-associated molecular networks and AFM-based quantification into one coherent picture, the review makes a compelling case that the adhesion gateway is where biology meets physics, and that learning to speak its language will define the next generation of therapies and engineered tissues.

Subject of Research: Mechanobiological signaling at cell adhesion sites and its role in cytoskeletal reconfiguration

Article Title: Adhesion gateway: decoding mechanobiological cues for cytoskeletal reconfiguration

Article References: Mukhopadhyay, G., Velu, K., Dhinakarasamy, I., Somu, P., Iyyappan, J., & Mani, R. (2026). Adhesion gateway: decoding mechanobiological cues for cytoskeletal reconfiguration. Molecular Biology Reports, 53(1), Article 1756. https://doi.org/10.1007/s11033-026-12912-6

Image Credits: AI Generated

DOI: 10.1007/s11033-026-12912-6

Keywords: mechanobiology, focal adhesion, integrins, cytoskeleton, mechanotransduction, talin, vinculin, YAP, substrate stiffness, atomic force microscopy, tissue engineering, cell adhesion

Collagen is the workhorse structural protein of the heart valve leaflets, providing the tensile strength that allows them to withstand the relentless mechanical demands of pumping blood. The fluid surrounding the heart contains collagenases that would, under ordinary circumstances, dismantle this critical protein scaffold. Yet healthy valve leaflets remain sturdy and functional for decades, opening and closing millions of times each year without losing their integrity. Understanding why has been a longstanding puzzle in cardiovascular biomechanics.

Lead author Hsiao-Ying Shadow Huang, an associate professor of mechanical and aerospace engineering at North Carolina State University, explained the motivation behind the research. The team knew that collagen was critical for keeping heart valves strong, and that the leaflets open and close millions of times every year. They also knew that the fluid surrounding the heart contains collagenases designed to break that collagen down, yet the leaflets remain sturdy for decades. When people develop heart disease, however, the structural integrity of those leaflets can degrade very quickly — and the researchers wanted to explore what role, if any, biomechanics might play in these contrasting outcomes.

To investigate, the researchers conducted experiments using pig hearts, whose valve anatomy closely resembles that of humans. They tested different levels of strain on heart valve leaflets, pulled the tissue in different directions, and varied the levels of collagenase in the fluid surrounding the hearts. The experimental design was carefully calibrated to replicate the natural mechanical environment of a beating heart while systematically altering key variables to isolate the protective mechanism.

The key insight came from understanding the precise geometry of leaflet deformation during each heartbeat. When a healthy heart pumps and the aortic valve leaflet is in the open position, the leaflet is stretched to 70 percent of its maximum capacity lengthwise but to only 20 percent of its maximum capacity laterally. The researchers tested the leaflets under those conditions but also examined other strain levels that retained that same 70-to-20 ratio, such as 35 percent to 10 percent. They also tried changing the ratio so that the leaflets experienced the same amount of strain in both directions. All of these experiments were conducted with both normal levels of collagenase and elevated levels of the enzyme.

After each experiment, the team evaluated the microstructure of the leaflet tissue to determine whether the collagen had remained intact or had been damaged. The results were striking. Increasing the strain did not damage the collagen, even with higher levels of collagenases, as long as the ratio of lengthwise strain to lateral strain stayed the same. But when the researchers changed the strain ratio, the collagen broke down — regardless of the collagenase levels present in the surrounding fluid.

This finding reveals that stretching the leaflet in the same direction as the collagen-rich fibers serves as a biomechanical protective mechanism, provided the naturally occurring strain ratio is preserved. The directional alignment of mechanical tension appears to physically shield the collagen molecules from enzymatic attack, effectively locking the protein into a configuration that collagenases cannot easily access or degrade. It is a remarkable example of how the body harnesses mechanical forces not just to move and pump, but to actively defend its own structural components from chemical breakdown.

Previous studies had identified this protective mechanism in collagen-rich tissues such as tendons, but that research only pulled the tissue in one direction. Heart valve leaflets, by contrast, experience strain in more than one direction every time they open and close, making the current finding a significant step forward in understanding how multiaxial mechanical loading protects soft tissues. The study, published in the Journal of Biomechanical Engineering under the title

The experimental system used in the study offers a useful window into how biomechanics research is actually conducted. Pig hearts are a common stand-in for human cardiac tissue because the dimensions, layered architecture, and collagen organization of porous valve leaflets closely mirror those found in people, which makes the findings plausibly transferable to human physiology. By isolating leaflets and subjecting them to controlled stretch in two perpendicular directions while varying the collagenase concentration in the surrounding fluid, the researchers could decouple mechanical loading from enzymatic exposure — two factors that are inseparable in a living heart.

The concept underlying the discovery is sometimes referred to in the literature as mechanical stabilization of collagen against proteolysis, meaning the breakdown of proteins by enzymes. Collagenases and related matrix-degrading enzymes normally access the triple-helical collagen molecule at specific sites along its length. When the molecule is held under tension along its fiber axis, those vulnerable regions can become less accessible, effectively hiding the degradation targets from the enzymes. The new work extends this idea to a more realistic loading scenario, because valve leaflets are stretched along their length and across their width simultaneously with every heartbeat, not in a single direction as in earlier tendon experiments.

The 70-to-20 strain ratio observed in the open aortic valve is not arbitrary. It reflects the natural architecture of the leaflet, in which collagen fibers are predominantly aligned in the circumferential direction, the direction that bears the greatest load when the valve is pressurized by blood. Stretching preferentially along that fiber direction appears to be what stabilizes the collagen, whereas distorting the ratio — for example, by stretching the tissue equally in both directions — disrupts the protective configuration and leaves the protein exposed to enzymatic attack regardless of how much collagenase is present.

This mechanism may help explain a broader clinical observation: structural valve deterioration tends to accelerate in settings where valve mechanics are altered. When heart disease changes the pressures and geometries inside the heart, the leaflets are deformed differently than they would be in a healthy beat, and the study suggests that this loss of the natural strain ratio strips away a biomechanical defense the tissue normally relies on. In such a state, even ordinary collagenase activity could produce damage far more rapidly than it would in a healthy valve.

The findings also carry implications for the engineering of replacement heart valves. Bioprosthetic valves, which are typically built from animal-derived tissue, eventually fail in part because their collagen degrades over time inside the body. If the protective effect of physiological strain ratios can be reproduced in engineered biomaterials — either by designing scaffolds that align collagen fibers appropriately or by conditioning implants to experience the same multiaxial loading patterns as native leaflets — it could extend the functional lifespan of valve replacements and reduce the need for repeat surgeries.

There are also broader lessons for connective tissue biology. Tendons, ligaments, skin, and blood vessel walls are all rich in collagen and all experience mechanical loading in daily life. The idea that tension along the fiber direction shields collagen from enzymatic breakdown suggests that mechanical environment is not merely a passive background condition but an active participant in tissue maintenance. Loss of appropriate loading — through immobilization, disease, or altered mechanics — may therefore expose collagen-rich tissues to faster degradation than their enzyme levels alone would predict.

The study, published in the Journal of Biomechanical Engineering, was co-authored by Siyao Huang, a former Ph.D. student at NC State, and Kaitlyn Barbour, a former master’s student at NC State. The work was an experimental study conducted in animal tissue, and the authors reported no competing interests. Because the experiments were performed on excised leaflets under controlled conditions rather than in living hearts, further work will be needed to confirm that the same strain-ratio-dependent protection operates continuously in the beating heart and to determine how the mechanism might be harnessed therapeutically. Even so, the results provide a concrete biomechanical account of a puzzle that has long puzzled cardiovascular scientists: how a tissue subjected to both relentless motion and a hostile enzymatic environment can remain intact for a lifetime.

Subject of Research: Biomechanical strain-stabilization of collagen in heart valve leaflets

Article Title: Study offers insights on how heart valves last so long

Article References: Study offers insights on how heart valves last so long. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: heart valves, collagen, biomechanics, collagenase, strain-stabilization, proteolysis, cardiovascular research, tissue engineering, Journal of Biomechanical Engineering, NC State University, offers, insights

Collagen is the most abundant protein in the human extracellular matrix, providing structural integrity to bones, skin, tendons, and virtually every connective tissue. What makes it scientifically extraordinary is its non-centrosymmetric triple-helical structure. Unlike materials whose molecular arrangement is symmetric in all directions, collagen’s helical architecture lacks a center of symmetry, which is the fundamental prerequisite for piezoelectricity. When mechanical force deforms the helix, the displacement of charged molecular groups within the structure creates a net electrical polarization. This means collagen can convert mechanical energy from body movement directly into localized electrical signals without any external power source, a property that has profound implications for how cells behave in their microenvironment.

The review, which surveyed literature from January 2000 to June 2026 across PubMed, Scopus, and Web of Science, identified that collagen-based piezoelectric biomaterials have shown promise across multiple tissue types, including bone, neural, skin, and cardiac tissue engineering. The key insight is that piezoelectric scaffolds can harness the body’s own endogenous mechanical stimuli, such as walking, breathing, or cardiac pulsation, and convert them into localized electrical cues at the implant site. These electrical signals influence cell adhesion, migration, differentiation, and extracellular matrix remodeling, all without the need for batteries, wires, or external stimulators. This self-powered approach represents a fundamental shift from traditional electrically active implants that require external energy.

At the nanoscale, the origins of collagen piezoelectricity have been progressively uncovered through advanced characterization techniques. Piezoresponse force microscopy has revealed nanoscale electromechanical heterogeneity within the subfibrillar structure of collagen fibrils, meaning that piezoelectric properties vary across the molecular building blocks of the protein. Molecular dynamics simulations have further clarified the polarization mechanisms within the super-twisted collagen structure, showing how the specific amino acid sequence and the spatial arrangement of alanine, hydroxyproline, and glycine residues contribute to the net piezoelectric response. These studies demonstrate that collagen’s piezoelectricity is not a uniform bulk property but emerges from the precise molecular geometry of its triple helix.

Fabrication strategies for collagen-based piezoelectric biomaterials have evolved considerably. Electrospinning, a technique that uses electrical forces to draw polymer solutions into nanofibers, has emerged as a leading method for creating collagen-containing piezoelectric scaffolds with controlled fiber alignment and porosity. Near-field electrospinning allows even greater precision in fiber placement, enabling researchers to pattern scaffolds that mimic the anisotropic architecture of native tissues. The alignment of electrospun fibers matters enormously, because the direction of fiber orientation determines the direction of piezoelectric response, which in turn influences how cells orient and migrate on the scaffold surface.

Hybrid systems that combine collagen with other piezoelectric or conductive materials have demonstrated enhanced electromechanical performance beyond what collagen alone can achieve. Barium titanate, a ceramic piezoelectric material with a high piezoelectric coefficient, has been incorporated into composite scaffolds to boost the overall electrical output. Conductive polymers have also been integrated to improve charge transport within the scaffold matrix. These hybrid approaches address one of the key limitations of pure collagen, which is that its piezoelectric coefficient is relatively modest compared to synthetic ceramics. By combining collagen’s biocompatibility and biodegradability with the superior piezoelectric performance of synthetic materials, researchers can create scaffolds that are both biologically compatible and electrically potent.

In bone tissue engineering, piezoelectric scaffolds have shown particular promise because bone itself is a naturally piezoelectric tissue. The review identified that piezoelectric signals play a role in vascularized bone regeneration, and that electrical stimulation can promote osteogenic differentiation of stem cells. Ultrasound-activated piezoelectric scaffolds have been developed to target specific intracellular signaling pathways, including calcium ion and NF-κB signaling, to orchestrate immunomodulation and osteogenesis for accelerated bone regeneration. The converse piezoelectric effect, where an applied electrical field produces mechanical deformation, has also been explored as a novel approach for applying mechanical stimulation to bone tissue without external loading.

In neural tissue engineering, electrospun piezoelectric scaffolds have been shown to support neurite extension of primary neurons, suggesting that the localized electrical cues generated by these materials can guide nerve regeneration. Biodegradable piezoelectric sensors have been developed for real-time evaluation of motor function recovery after nerve injury, demonstrating that the same materials used for tissue regeneration can also serve as diagnostic tools. In skin wound healing, piezoelectric film scaffolds have promoted healing with enhanced collagen deposition and blood vessel regeneration through upregulation of the PI3K/AKT signaling pathway. Three-dimensional printed piezoelectric wound dressings have been developed with dual piezoelectric response models for scar-prevention wound healing, and self-powered piezoelectric nanofibrous hydrogels have been designed for synergistic electro-pharmacological therapy of chronic wounds.

Beyond tissue regeneration, the review highlights the emerging potential of collagen-based piezoelectric materials for biosensing and energy harvesting applications. Piezoelectric nanogenerators can convert human motion into electrical energy for powering wearable and implantable devices, and self-powered sensors based on these materials can monitor physiological signals in real time without batteries. Collagen-gold nanoparticle conjugates have been developed for versatile biosensing, and collagen-based biopiezoelectric nanogenerators have been prepared from biological waste materials such as fish skin, demonstrating the sustainability potential of these approaches. Flexible pressure sensors, wearable patches for sweat analysis, and implantable BioMEMS devices all represent potential integration points for collagen-based piezoelectric platforms in next-generation healthcare systems.

Despite the remarkable progress, the review emphasizes that clinical translation of collagen-based piezoelectric biomaterials requires further advancement in several critical areas. Performance variability remains a challenge, as the piezoelectric properties of collagen can differ depending on source, processing method, and environmental conditions. Degradation stability must be improved to ensure that scaffolds maintain their electromechanical performance throughout the tissue regeneration process. Seamless integration with flexible electronic systems is essential for combining tissue engineering with biosensing functionality. The review concludes that standardized fabrication protocols, rigorous in vivo validation, and careful consideration of regulatory requirements will be essential for advancing these materials toward practical biomedical use. As research continues to unravel the molecular mechanisms of collagen piezoelectricity and refine fabrication strategies, these materials stand poised to redefine the interface between biology and electricity in medicine.

Subject of Research: Bio-piezoelectric mechanisms and biomedical applications of collagen-based piezoelectric biomaterials

Article Title: Bio-Piezoelectricity of Collagen: Mechanisms, Tissue Engineering Applications, and Biosensing Potential

Article References: Pan, C.-T., Laya, S., Shiue, Y.-L., & Paudel, T. (2026). Bio-Piezoelectricity of Collagen: Mechanisms, Tissue Engineering Applications, and Biosensing Potential. Journal of Medical and Biological Engineering. https://doi.org/10.1007/s40846-026-01059-1

Image Credits: AI Generated

DOI: 10.1007/s40846-026-01059-1

Keywords: collagen, piezoelectricity, tissue engineering, regenerative medicine, biosensors, electrospinning, nanogenerators, biomaterials, bone regeneration, wound healing, energy harvesting, extracellular matrix

The challenge the researchers faced is fundamental to the biology of the cells themselves. Adipocytes store most of their volume as a single large lipid droplet, which makes them less dense than the aqueous media surrounding them. In a liquid or semi-liquid bioink, they float, drift, and cluster rather than staying where the printer places them. Their hydrophobic surfaces and large size — often 50 to 100 micrometers across — mean that standard extrusion through narrow nozzles tends to rupture their membranes. Most existing 3D adipose models sidestep the problem by using adipose-derived stem cells that are coaxed into becoming fat cells after printing. That approach works, but it produces less mature tissue with different signaling behavior. Fully differentiated adipocytes are more physiologically faithful, which is why the Stuttgart team chose to print them directly, despite the mechanical risks.

Their solution combines two gels with complementary roles. The cells are suspended in a low-concentration gellan gum bioink, a polysaccharide gel that is fluid enough to be extruded gently but can be stabilized after deposition by adding calcium chloride, which crosslinks the polymer chains. The printed material is not deposited into open air, where a soft bioink would simply slump and spread. Instead, it is extruded into a chilled agarose support bath — a self-standing gel that holds each filament in place the moment it leaves the nozzle. This technique, sometimes called embedded or freeform bioprinting, counteracts both the spreading of the ink and the buoyancy of the cells, allowing accurate layer-by-layer construction of soft structures that would collapse under conventional printing.

To find a formulation that balanced fluidity with stability, the researchers isolated mature adipocytes from human adipose tissue obtained during elective surgery and mixed them into gellan gum at a final polymer concentration of 0.3 percent. They tested formulations containing different concentrations of phosphate-buffered saline and measured the viscosity of both the bioink and a 0.5 percent agarose support bath across a range of shear rates. The goal was a bioink that would flow smoothly under gentle pressure yet hold its shape once crosslinked, and a support bath that would remain solid enough to confine the printed filaments without crushing them. The optimization identified a combination that satisfied both requirements, allowing the team to extrude the cell-laden ink through a 600-micrometer nozzle at pressures of 4 to 6 kilopascals and speeds of 14 to 16 millimeters per second — conditions mild enough to spare the delicate cells.

The structural results were striking. When the same soft bioink was printed without a support bath, it spread after extrusion and could not be layered reliably into defined shapes. With the agarose bath, the team produced stable grid structures measuring 20 by 20 by 1 millimeters with uniform pores and well-aligned filaments. Quantitative comparison against the computer-aided design showed remarkably low deviation: construct length differed by only 2.66 percent, pore width by 2.87 percent, and filament width by 3.35 percent. For a material soft enough to mimic adipose tissue — which is among the softest tissues in the body — that level of geometric fidelity is a meaningful technical achievement, since it demonstrates that printing accuracy and physiological softness need not be traded against each other.

Cell survival was the second critical test. Live and dead staining showed that most adipocytes remained viable through three days after printing, and viable cells were still observed on day eight in an exploratory assessment. One telling indicator came from measurements of lactate dehydrogenase, an enzyme that leaks out of cells when their membranes are damaged. Release dropped significantly from 29.09 percent on day one to 9.66 percent on day three, suggesting that the transient membrane stress caused by printing was real but recoverable — the cells repaired themselves once settled into their new gel environment. This pattern matters because it distinguishes a gentle process that temporarily perturbs cells from a harsh one that permanently destroys them.

Morphology and function held up as well. Lipid staining revealed uniformly distributed, lipid-filled cells throughout the constructs, and expression of perilipin A — a protein that coats lipid droplets and is a standard marker of intact adipocyte structure — confirmed that the cells’ characteristic unilocular architecture had been preserved. Most importantly for anyone hoping to model metabolism, the printed cells still responded to hormonal stimulation. When the researchers applied isoproterenol, a beta-adrenergic agonist that triggers lipolysis, the cells released glycerol in dose-appropriate fashion: from 40.60 to 137.88 nanograms per milliliter on day one, and from 65.86 to 200.54 nanograms per milliliter on day three. In other words, the printed fat tissue could still perform its core metabolic job of breaking down stored lipid on command.

Why does this matter beyond the printing bench? Adipose tissue is not merely passive storage; it is a metabolically active organ involved in energy storage, endocrine signaling, and whole-body metabolic regulation. Dysfunction of adipocytes contributes to obesity, insulin resistance, type 2 diabetes, and other diseases, which creates strong demand for human-relevant laboratory models. Two-dimensional cultures cannot reproduce the soft mechanical environment, spatial organization, or buoyancy effects of native fat tissue, while animal models suffer from species differences as well as ethical and financial constraints. A 3D construct built from genuine mature human adipocytes, held in a defined geometry, closes much of that gap and could support preliminary drug and cosmetic testing before animal or clinical studies.

The Stuttgart team is candid about the limits of the current work. The culture periods examined were short, and real adipose tissue depends on interactions with endothelial cells, immune cells, and stromal cells, as well as on a functioning vasculature that supplies oxygen to cells living deep within tissue. Extending culture duration, introducing vascularization, and incorporating those additional cell types are the obvious next steps. The support-bath method, however, provides a practical foundation on which such complexity can be built, because it solves the hardest problem first: keeping mature adipocytes alive and functional while placing them exactly where a design requires.

The broader significance lies in accessibility. The workflow relies on relatively inexpensive materials — gellan gum and agarose are common, food-grade polysaccharides — and standard extrusion bioprinting equipment, making the approach reproducible across laboratories without exotic hardware. If follow-up studies confirm the method at longer time scales and in more complex, vascularized constructs, printed human fat tissue could become a routine platform for studying metabolic disease, screening compounds that affect fat cell function, and eventually engineering soft tissue for regenerative and reconstructive medicine. For a cell type long considered too fragile to print, the fat cell has just become considerably more approachable.

Subject of Research: Support-bath bioprinting of mature human adipocytes in gellan gum hydrogels for functional 3D adipose tissue models

Article Title: Support-bath bioprinting creates functional 3D human fat cell models

Article References: Support-bath bioprinting creates functional 3D human fat cell models. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: bioprinting, adipocytes, support bath, gellan gum, agarose, 3D tissue models, obesity, metabolic disease, adipose tissue, tissue engineering, lipolysis, gellan gum bioink