Bioactive Materials

Disruption of the wound healing cascade can result in pathological outcomes, including fibrosis due to myofibroblast-mediated contraction and collagen deposition. Despite the clinical significance, effective treatments for fibrosis remain limited as current therapies often show inconsistent efficacy, adverse effects, and patient discomfort. Combinatorial therapeutic strategies integrating biomaterial scaffolds with gene delivery have shown promise in regenerative healing. MicroRNAs (miRNAs) are key regulators of fibrotic signalling in cells, including fibroblasts and myofibroblasts. Specifically, miRNA-29b is notable for downregulating pro-fibrotic genes, including collagen type I, reducing ECM accumulation, and limiting fibroblast/myofibroblast overactivation. In this context, the present work develops a collagen-GAG (CG) scaffold platform for delivery of miRNA-29b complexed GET nanoparticles to inhibit fibrosis. Initially, bioinformatic analysis of miRNA-29b validated its involvement in ECM-associated pathways and processes, followed by successful nanoparticle internationalisation in primary dermal fibroblasts. The anti-fibrotic efficacy of the optimised miRNA-29b nanoparticles was subsequently demonstrated by significant reductions in collagen deposition and -SMA expression, both key indicators of myofibroblast differentiation and fibrosis. The optimised miRNA-29b formulation was then incorporated into 3D porous collagen-GAG (CG) scaffolds, which modulated fibrotic gene expression while preserving scaffold structure conducive to fibroblast/myofibroblast infiltration and proliferation. Finally, functional outcomes of seeded TGF-{beta}-stimulated fibroblasts, including reduced matrix contraction, -SMA expression, and ECM deposition, were comparable to those observed in non-fibrotic conditions, thereby confirming the therapeutic potential of scaffold-mediated miRNA-29b delivery. Together, these findings demonstrate that scaffold-mediated miRNA-29b delivery represents a promising anti-fibrotic strategy for wound healing by mitigating myofibroblast activation, limiting matrix contraction, and preventing pathological ECM accumulation.

Chronic diabetic wounds resist healing due to impaired angiogenesis, stalled cellular migration, and persistent inflammation, a challenge further compounded by the rapid degradation of therapeutic growth factors in the proteolytic wound bed. To overcome this, Traditional Chinese Medicine (TCM) compounds are employed not as standalone drugs, but as biomolecular stimuli to precondition the secretome of Wharton's Jelly-derived mesenchymal stem cells (WJMSCs). To overcome these critical translational barriers, this study engineers a core-shell coaxial electrospun nanofibrous scaffold (polyvinyl alcohol core/gelatin shell) designed for the stabilizing and sustained dual-delivery of biologics. We introduce a novel synergistic payload with WJMSCs conditioned medium (WJMSCs-CM) uniquely primed by two specific chinese herbal compounds, Astragaloside IV (AS-IV) and Formononetin (FMN). This core-shell architecture provides native-like contact guidance for cells while converting conventional burst release into a sustained, weeks-long elution. In vitro, this functionalized scaffold restores Akt/eNOS signaling, rescues cellular viability, and promotes robust tube formation in high-glucose-stressed fibroblasts and endothelial cells. In vivo, within an STZ-induced diabetic rat model, the application of this WJMSCs-CM-loaded coaxial scaffold actively inhibits early inflammation and comprehensively accelerates healing, driving near-complete wound closure (98.2 by day 21), mature collagen deposition, and hair follicle neogenesis. Ultimately, this bio-instructive platform successfully integrates physical structural cues with sustained biochemical signaling, offering a potent, multifaceted strategy for chronic wound regeneration.

For regenerative medicine applications, transplanted cells are difficult to retain in a specific place to maximize the localized release of cytokines, which can positively modulate immune responses, angiogenesis and tissue regeneration. Here, we developed a hollow electrospun (ESP) scaffold with one open and one closed end to enclose a hydrogel laden with human mesenchymal stem cells (hMSCs); we termed this combination the hydrocup. hMSCs remained viable for 28 days in vitro and released functional cytokines similar to hMSCs in transplanted but non-enclosed hydrogels. The hydrocups remained intact and fixed in place for 6 weeks after subcutaneous implantation in rats, demonstrating in vivo compatibility. These results demonstrate that the hydrocup is the first, to our knowledge, cell-laden hydrogel delivery scaffold. This innovation could be developed for a wide variety of cell-based cytokine secretion or drug-laden hydrogel applications.

Background: Post-operative uterine adhesions remain a major clinical challenge, often leading to infertility and severe pelvic pain. Current anti-adhesion barriers lack the mechanical strength, tissue specificity, or bioactivity required for optimal regeneration of uterine tissue. To address these limitations, we developed a biomimetic, multilayer scaffold that replicates the structural and functional complexity of the myometrial and serosal layers of the uterus by combining a tunable polycaprolactone (PCL) mesh with a bioactive hydrogel derived from decellularized uterine extracellular matrix (dUECM) reinforced with sodium alginate (Alg). Materials and Methods: Composite scaffolds were fabricated using near-field melt electrowriting (MEW) to create precision-engineered PCL meshes with various infill angles (90{degrees}, 60{degrees}, 45{degrees}, and 30{degrees}), followed by impregnation with an alginate-dUECM hydrogel. A comprehensive physicochemical characterization was conducted using Fourier Transform Infrared Spectroscopy (FTIR), Thermogravimetric Analysis (TGA), and contact angle measurements. Mechanical properties were evaluated in both dry and wet states via uniaxial tensile testing. Swelling and degradation were assessed over 10 days. hTERT-HM cells were used for in vitro evaluation through Live/Dead staining, MTT, SEM, and immunocytochemistry for -SMA and DAPI. Results: The successful incorporation of the hydrogel into the PCL mesh was confirmed by the results obtained from FTIR and TGA analyses; these findings were further supported by contact angle studies, which showed a marked increase in hydrophilicity, resulting in a reduction of the static contact angle from 113 {+/-} 4{degrees} in the pure PCL to 60 {+/-} 12{degrees} in the hydrogel-infiltrated samples in the 30{degrees} group. Mechanical testing showed that the wet composite scaffolds maintained higher stiffness than hydrated native uterine tissue. Cell culture assays confirmed excellent cytocompatibility and proliferation, especially in 30 infill architecture. Immunofluorescence revealed strong -SMA expression and cytoskeletal organization, indicating phenotypic maturation of uterine smooth muscle cells for all groups. Conclusion: Among all groups, the 30 MEW mesh impregnated with Alg-dUECM hydrogel demonstrated the most balanced combination of mechanical resilience, degradation profile, wettability, and cellular compatibility. This structure most closely recapitulates the biological characteristics of uterine outer myometrium tissue, positioning it as a highly promising scaffold for regeneration and biofunctional anti-adhesion barrier applications.

Recent efforts to reconstruct critical-sized craniomaxillofacial defects using biomaterials have increasingly sought strategies to accelerate proangiogenic processes immediately after surgical implantation to improve host-graft integration. A class of mineralized collagen scaffolds has recently been identified to promote osteogenesis of bone marrow and adipose-derived mesenchymal stem cells both in vivo and in vitro, motivating efforts to identify strategies to promote osteogenic-angiogenic coupling in order to support regenerative healing. Here, we report the first-time use of a glycoengineering approach to generate a population of engineered ASCs (eASCs) from human adipose-derived stem cells (hASCs). This effort inserts an azido sugar onto the cell membrane that can be selectively targeted using DBCO-tagged biomolecules to selectively boost eASCs activity. We report the effects of azido sugar labeling conditions (concentration and incubation time) on eASCs labeling efficiency. We show proliferation and pro-regenerative gene expression activity of eASCs in mineralized collagen scaffolds are largely similar to conventional hASCs. And we demonstrate that molecular cargo functionalized with dibenzocyclooctyne (DBCO) can be conjugated to azido groups on eASCs via click chemistry. Together, this work shows metabolically labeled eASCs have the potential to be used for selective delivery of activity-inducing bioactive cues to enhance regenerative potential.

Tympanic membrane perforations remain a common clinical problem, and although surgical intervention through tympanoplasty achieves high success rates, it is associated with donor-site morbidity, surgical complexity and limited restoration of the native radial and circumferential collagen architecture. In this study, 3D extrusion printing was utilized to create an active scaffold and attempt to promote collagen organization through shear-mediated structural alignment. An alginate-carboxymethyl cellulose (CMC) hydrogel with bovine SIS-dECM was prepared and investigated for its suitability as a bioink alternative to tympanoplasty grafts. The physiochemical, rheological and printability characteristics of the hydrogel were assessed. Successful decellularization was confirmed by histological analysis. The incorporation of the SIS-dECM into the hydrogel led to increased swelling, lower apparent viscosity, yield stress and flow stress while maintaining favourable printability and filament stability. Polarized optical microscopy was also used to study the influence of printing speed on the alignment of collagen to mimic the native tympanic membrane radial collagen architecture. Compared with the cast controls, the printed samples presented stronger birefringence signals. Biological evaluation demonstrated that the 15% dECM hydrogel exhibited the highest live cell area percentage and live/dead ratio after 48 h. In addition, the chick chorioallantoic membrane assay demonstrated that the dECM-containing hydrogels improved vascular density. The findings establish a printable, biologically active dECM bioink capable of generating bulk collagen organization through extrusion printing as a platform for tympanic membrane regeneration.

Articular cartilage damage often progresses to osteoarthritis (OA), a degenerative joint disease characterized by chronic pain, limited mobility, and reduced quality of life. Tissue engineering approaches using poly (ethylene glycol) (PEG)-based hydrogels offer tunable mechanical properties and bioactive functionalization, yet the influence of surface charge on cartilage regeneration remains underexplored. Moreover, recombinant fibroblast growth factor 18 (FGF18) has successfully improved cartilage tissue thickness in clinical trials, but required high dosages and recurring injections may limit compliance. Here, we developed a granular microgel-based platform with bioorthogonally tethered FGF18 to evaluate the interplay of microgel surface charge and growth factor presentation on chondrogenesis. Azide groups were incorporated onto the microgel surfaces to enable site specific FGF18 conjugation across microgel scaffolds with distinct surface charges. When seeded with mesenchymal stromal cells, microgel scaffolds functionalized with FGF18 outperformed their unmodified counterparts, evidenced by higher GAG content, collagen content, and compressive modulus. In a murine microfracture model, anionic and zwitterionic microgel scaffolds tethered with FGF18 increased cartilage regeneration compared to nonionic microgels. We detected increased collagen II content within defects treated with tethered FGF18 microgel scaffolds. This work demonstrates the role of surface charge and growth factor presentation in directing cell behavior and tissue repair, advancing the design of biomaterials for cartilage regeneration. HIGHLIGHTSO_LICovalent tethering of FGF18 to microgel surface enables localized bioactivity C_LIO_LIFGF18 tethered microgel scaffolds enhance extracellular matrix deposition and chondrogenic differentiation of murine mesenchymal stromal cells in vitro C_LIO_LILocalized FGF18 presentation improves cartilage tissue formation and mechanical properties in vivo, with anionic and zwitterionic microgel scaffolds outperforming nonionic scaffolds C_LIO_LIFGF18 presentation is a more potent stimulus than microgel surface charge for cartilage regeneration C_LI

Chronic diabetic wounds remain a major clinical challenge because current therapies address infection or supplement growth factors without correcting the cellular dysfunction that prevents regeneration. We identify pathological glycocalyx thickening in diabetic dermal fibroblasts (DDFs) as a driver of elevated caspase 3, 8, and 9 expression and heightened apoptosis -- deficits that stall wound repair. Cleavage of sialic acid residues by neuraminidase (NMase) reverses this dysfunction, restoring fibroblast migration, proliferation, and contractility. We engineer a photo-crosslinked hybrid hydrogel combining methacrylated gelatin (GelMA) with high-molecular-weight methacrylated chitosan (HMW ChMA). ChMA increases storage modulus 8-12-fold, reduces pore size, and confers antibacterial activity against gram-positive and gram-negative bacteria, addressing infection susceptibility. The fortified HMW hybrid (HMWH) network enables sustained, localized NMase delivery that outperforms GelMA alone in resisting degradation and controlling release kinetics. NMase-loaded HMWH (N-HMWH) gels enhance DDF proliferation and migration in vitro, correlating with reduced focal adhesion size and increased turnover. In a diabetic rat model, N-HMWH patches achieve superior wound closure, outperforming EGF therapy, with robust epidermal regeneration, neovascularization, and collagen deposition. This work establishes glycocalyx-targeting hydrogels as a new class of wound therapeutics addressing the root cause of diabetic fibroblast failure, not just compensating with growth factors.

Neural progenitor-stem cells (NPSCs) reside in mechanically dynamic brain microenvironments and give rise to neurons, astrocytes, and oligodendrocytes. Although recent studies have shown that matrix viscoelasticity can influence neural maturation and neurogenic differentiation, its role in primary NPSCs beyond neurogenesis remains less defined. How matrix viscoelasticity or stress relaxation regulates primary NPSC stemness, neuronal and glial differentiation, and the associated matrix-cell mechanotransduction pathways are not well understood. Here, we use alginate hydrogels with independently tunable stiffness and stress relaxation properties to investigate how matrix stress relaxation regulates the fate of primary subventricular zone (SVZ)-derived NPSCs in 3D. The results suggested that matrices with faster stress relaxation enhances stemness maintenance, radial glial-like marker expression, and differentiation of NPSCs toward neuronal, astrocytic, and oligodendrocytic lineages in the corresponding biochemical environments. In mixed neuronal/astrocytic differentiation conditions, fast-relaxing matrices preferentially promote neuronal differentiation. Mechanistically, NPSC responses to matrix stress relaxation involve integrin-mediated adhesion, actomyosin contractility, actin polymerization, and Piezo1 activity, with distinct contributions across differentiation lineages. Together, these findings reveal how matrix stress relaxation regulates primary NPSC stemness and multilineage differentiation through multiple mechanotransduction pathways in 3D.

The cornea is the outermost layer of the eye, most vulnerable to injury including from chemical exposure, either environmental, industrial or chemical weapons. For example, exposure of the eye to sulfur mustard (SM) can cause mustard gas keratopathy, resulting in devastating consequences for vision. Available treatments for corneal mustard keratopathy are either ineffective, or if used for a prolonged time, have deleterious side effects. Here, we aimed to harness the anti-inflammatory and anti-angiogenic peptide angiotensin-(1-7), known as Ang-(1-7), that is derived from the renin-angiotensin system (RAS). We designed and developed a protein-like polymer (PLP) displaying a brush of norleucine modified Ang (1-7) peptides (NLE Ang (1-7) PLP), which is a more potent derivative of Ang-(1-7), for administration as an eye drop. In a mouse model of nitrogen mustard (NM)-induced corneal injury, topical treatment and intraperitoneal (i.p.) injection of NLE Ang (1-7) PLP persistently minimized: (i) corneal haze; (ii) edema; (iii), expression of pro-inflammatory and pro-angiogenic genes; and (iv) neovascularization and conjunctivalization as evidenced after a prolonged treatment and recovery period. Collectively, our observations have demonstrated the efficacy of this novel polymer proteomimetic based eye drop treatment in reducing chemical agent-induced corneal injury as well as indicating its potential application in other ocular surface diseases associated with inflammation and angiogenesis.

Damage to the skin by trauma, burns, or surgical procedures often results in uncontrolled bleeding, which remains a leading cause of preventable death following injury, yet most conventional hemostatic materials are engineered solely to arrest bleeding and often adhere strongly to the wound bed, causing pain, rebleeding, and disruption of newly formed tissue upon removal. Here, we report a DNA hydrogel that structurally mimics neutrophil extracellular traps (NETs) and is crosslinked using a bioactive small molecule with potent autophagy-inducing, cardioprotective, anti-inflammatory, antioxidant, and mitochondria-protective properties, integrating rapid hemostasis with active support for tissue regeneration in a single biomaterial. The DNA network provides an intrinsically biocompatible, biodegradable scaffold capable of recruiting platelets and erythrocytes to achieve rapid clot formation, while the bioactive crosslinker is released as the network degrades, delivering a sustained cytoprotective and anti-inflammatory stimulus directly at the wound site. The hydrogel was characterised physiochemically and evaluated for cytocompatibility, hemolytic potential, hemostatic efficacy, and wound-healing performance in a murine model. Results demonstrate that the bioactive-crosslinked DNA hydrogel achieves rapid, effective hemostasis, while accelerating wound closure and supporting regenerative tissue remodelling. This dual-function platform offers a promising strategy for next-generation wound-care biomaterials that unite immediate bleeding control with accelerated, natural tissue healing.

Engineering a transplantable artificial liver requires reconstituting the hepatic lobule, whose densely cellular parenchyma is organized around a hierarchically branched vascular network. Achieving high cellular density and hierarchical vascularization within a single fabrication step remains a major challenge. Here, we present an ultrasonic standing wave (USW)-based strategy that concurrently organizes vascular and parenchymal compartments during a single extrusion. HepG2-endothelial cell aggregates were fabricated as building blocks and suspended in a liver-derived dECM-alginate hydrogel for rapid, ultraviolet-free crosslinking. They were then co-extruded with single endothelial cells through a glass capillary under USW actuation. Due to their size difference, the single cells were focused at the pressure nodes, forming patterns that transitioned from a single supplying line to four microvascular lines. The aggregates instead accumulated at the pressure antinodes, reconstituting the basic microvascular-parenchymal unit of the lobule. Aggregate size increased with seeding density while maintaining uniformity and over 90% viability. The patterned endothelial cells formed perfusable lumina through which 5 um microparticles flowed freely. Compared with hepatocyte-only cultures, the vascularized constructs showed a 1.7-fold increase in urea production, and 2.7- and 6.2-fold increases in CYP1A2 and CYP3A4 expression, respectively. Following implantation into the mouse liver, the scaffolds showed favorable biocompatibility, sustained proliferation over 21 days, and progressive host integration. The smallest blood-containing lumina matured from 26.1 um to 7.9 um in diameter, accompanied by increasing collagen deposition at the graft-host interface. This approach provides a template-free route to hierarchically vascularized hepatic units and a promising step towards implantable liver tissue.

Intranasal administration provides a noninvasive route for local and systemic drug delivery, yet its clinical effectiveness remains limited by rapid mucociliary clearance and short drug residence times within the nasal cavity. Existing stimulus-responsive nasal hydrogels seek to address this challenge but rely on physiological triggers such as temperature, pH, or ionic strength to induce gelation, resulting in variable performance due to natural differences in the nasal cavity. Here, we present a polyethylene glycol (PEG)-based hydrogel platform that overcomes these limitations through rapid in situ gel formation that is independent of physiological stimuli. The two liquid precursor solutions remain sprayable during administration and rapidly crosslink upon deposition to form a cohesive hydrogel depot. Although this dual-component design presents challenges for compatibility with conventional nasal spray devices, we demonstrate successful delivery using commercially available dual-barrel spray systems. The hydrogel maintained spray performance and regional nasal deposition comparable to conventional aqueous formulations while significantly reducing mucociliary transport to prolong mucosal residence. Importantly, reduced mucociliary transport was achieved without impairing ciliary function or compromising epithelial barrier integrity, addressing key safety considerations for repeated intranasal administration. In addition, the hydrogel altered epithelial uptake and transport of biologics, suggesting its potential to improve the nasal delivery of macromolecular therapeutics that typically exhibit poor bioavailability. The results of these study establish a versatile and clinically translatable approach for enhancing intranasal drug delivery across a broad range of therapeutic applications.

The growing demand for three-dimensional (3D) in vitro models that accurately replicate human tissue to investigate disease mechanisms, enable tissue replacement, and support drug testing is driving the need for more advanced manufacturing platforms. Mesenchymal stem/stromal cells (MSCs) are promising candidates for tissue engineering and stem cell therapies due to their regenerative potential and ethical advantages. However, the reproducible and scalable production of 3D models that truly mimic the human complexity and function poses a major challenge. To address these challenges, we have developed a scalable and semi-automated 3D differentiation process for generating osteogenic microtissue derived from MSCs encapsulated in core-shell capsules (CSCs). To validate material-dependent, osteo-inductive effects of hyaluronic acid (HA), we systematically characterised our encapsulation process with regard to CSC integrity, spheroid formation and viability, as well as diffusion and shear thinning properties. Osteogenic differentiation was performed in static and dynamic culture, using either conventional 6-well plate format or a rotating wall vessel bioreactor. HA-CSCs were compared with inert carboxymethyl cellulose (CMC)-CSCs. In addition, hypoxic culture condition and the implementation of human platelet lysate (hPL) further supported physiological relevance. HA led to the formation of larger spheroids with high cell viability and increased metabolic activity compared to CMC. Calcium phosphate deposition within the extracellular matrix (ECM) and elevated ALP activity confirmed successful osteogenesis in all conditions. Furthermore, both HA and dynamic culture promote osteogenesis, and when combined, synergistically enhance the osteogenic differentiation. In conclusion, we present a semi-automated, high-throughput and xeno-free platform process for the 3D differentiation of MSCs towards osteogenic lineage. By integrating HA, hypoxia and dynamic culture conditions, this capsule-based system enhances the physiological relevance, enabling advanced in vitro disease modelling, drug testing, and scalable microtissue generation while reducing reliance on animal models.

Precise control of growth factor delivery remains a challenge for directing stem cell differentiation in three-dimensional (3D) engineered tissues. In this study, engineered cell-like vesicles are used as programmable microenvironments to enable sustained and localized delivery of growth factors within visible-light-crosslinked GelMA hydrogels. Giant unilamellar vesicles (GUV) loaded with FGF-2 and TGF-{beta}3 were incorporated into bioinks with BM-MSC to drive keratocyte differentiation without repeated soluble growth factor supplementation. ELISA measurements confirmed the removal of non-encapsulated growth factors and the release of the vesicle cargo following induced vesicle rupture. Fluorescence monitoring showed a progressive reduction in detectable FGF-2- and TGF-{beta}3-loaded GUV during culture, while droplet-scale analysis demonstrated the co-deposition of cells and vesicles after printing. After 14 days of differentiation, differentiated cells expressed ALDH1A1, ALDH3A1, lumican, keratocan, and collagen I without induction of -SMA. Interestingly, keratocyte-associated differentiation was retained after drop-on-demand bioprinting, confirmed by qPCR analysis. These findings establish growth factor-loaded vesicles as bioprintable instructive niches capable of supporting localized keratocyte differentiation within 3D corneal constructs.

Photocrosslinkable gelatin derivatives are promising bioinks for soft tissue engineering, but their use in cell-laden digital light processing (DLP) requires a balance between tunable mechanics, printability and cytocompatibility. Here, we report a small library of photocrosslinkable gelatin-based precursor formulations for cell-laden DLP, with the broader aim of generating a versatile platform for soft tissue engineering rather than a formulation restricted to one tissue model. A bifunctional methacryloyl-norbornene gelatin (GelMANB) was developed to combine methacryloyl chain-growth polymerisation with norbornene-mediated thiol-ene crosslinking and was benchmarked against gelatin methacryloyl (GelMA). GelMANB formulations containing different thiolated crosslinkers were evaluated to determine the influence of crosslinking chemistry as well as polymer concentration on network formation and physico-chemical properties. Thiol-ene formulations enabled broad tuning of hydrogel stiffness and exhibited rapid photocrosslinking, while crosslinker selection further influenced swelling and tensile behaviour. Selected GelMANB/dithiothreitol (GelMANB/DTT) and GelMANB/thiolated gelatin (GelMANB/GelSH) formulations were subsequently processed via DLP alongside GelMA. Optimised exposure conditions enabled reproducible fabrication of porous scaffolds that retained dimensional stability under osmotic pressures relevant to several soft tissues. Following cell-laden printing, human foreskin fibroblasts remained viable after seven days, reaching 95.0{+/-}5.9% viability in GelMANB/DTT and 94.9{+/-}2.7% in GelMANB/GelSH, compared with 87.2{+/-}6.0% in GelMA. In 2-mm-thick hydrogel discs, GelMANB/GelSH supported the most sustained fibroblast elongation, reaching 30.9{+/-}6.3% elongated cells at day 7, although elongated cells were mainly confined to the outer surface layers. Porous scaffolds with 400 m struts and 1.3 mm pores reduced the characteristic hydrogel thickness and enhanced elongated morphology across all formulations, highlighting GelMANB as a modular platform for cell-laden soft tissue biofabrication.

Collagen, a major component of the extracellular matrix, regulates cellular behaviors, such as adhesion, differentiation, and angiogenesis. These functions are mediated by interactions between specific amino acid motifs within the collagen triple-helical structure and collagen-binding biomolecules. These include cell-surface receptors, such as integrins, discoidin domain receptors (DDRs), and syndecans, a family of transmembrane heparan sulfate proteoglycans (HSPGs). Signals mediated by these receptors are integrated to regulate cell fate. However, native collagen simultaneously presents multiple receptor-binding motifs, making it difficult to isolate receptor-specific functions and to evaluate receptor crosstalk. Here, we introduce a composition-controlled artificial collagen matrix platform that enables independent tuning of multiple receptor-binding motifs within a constant triple-helical scaffold. This material was produced by disulfide crosslinking of chemically synthesized collagen-like triple-helical peptides, each bearing a single defined receptor-binding sequence. By varying the mixing ratios of these peptides before crosslinking, we systematically controlled the composition of receptor-binding motifs within the matrices. We applied this platform to nerve growth factor-dependent neuronal differentiation of PC12 cells, a process supported by collagen. Matrices containing only integrin-binding sequences were sufficient to support this differentiation. Incorporation of an HSPG-binding sequence had little additional effect, whereas incorporation of a DDR-binding sequence suppressed integrin-mediated differentiation and coincided with DDR phosphorylation. These results reveal opposing roles of collagen-binding integrins and DDRs in regulating PC12 cell differentiation. Composition-controlled artificial collagen provides a versatile matrix platform for dissecting functional crosstalk among collagen receptors.

Engineering biomimetic tissues with dynamically evolving 3D architectures represents an important direction for next-generation tissue engineering, as it enables recapitulation of the continuous morphogenesis of native tissues during development and regeneration. Here, a self-actuating 4D cell-strand bioprinting platform is developed to engineer complex tissue architectures through autonomous cell contractile force (CCF)-driven morphing without requiring external stimuli. The platform integrates a mechanically compliant and self-softening base hydrogel with embedded high-density cell strands printed using a fast-degrading carrier bioink. During culture, the carrier bioink rapidly degrades while the encapsulated cells proliferate and establish connected cellular networks, generating localized contraction that drives programmable shape transformation. Through spatial patterning of embedded cell strands, constructs with diverse morphologies, including V-shaped, helical, folded, and tubular architectures, are generated via controllable self-actuated morphogenesis. The platform further enables engineering of cartilage-like and bone-like tissues with well-defined curvature configurations and mechanically robust tissue matrices. In addition, programmable multi-tissue engineering is demonstrated through fabrication of a muscle-tendon junction-mimicking construct containing spatially organized fibroblast and myoblast compartments. This self-actuating 4D bioprinting strategy enables highly programmable and directionally controlled morphogenesis using a simple construct design with low cell amount requirements, providing a versatile platform for engineering dynamic tissue architectures.

Introduction: Advanced biomaterials for regenerative medicine are increasingly expected to combine multifunctionality and compatibility with tissue-specific cellular processes. In this context, hydroxyapatite-based platforms modified through ionic substitution represent promising candidates, as they may integrate structural similarity to bone mineral with additional biological functionality and luminescent properties, enabling diagnostic applications and real-time monitoring. In this study, we evaluated whether silicate-phosphate substituted calcium hydroxyapatite Ca10(PO4)6-x(SiO4)x(OH)2 (where x = 1.5) co-doped with lithium(I), europium(III), and gadolinium(III) ions (Si-HAp-LEG) affects the osteogenic, chondrogenic, and adipogenic differentiation potential of human bone marrow stromal/stem cells (BMSCs). Methods: Human BMSCs were cultured under lineage-specific differentiation conditions in the presence of undoped silicate-substituted phosphate hydroxyapatite (abbr. as Si-HAp), which served as a control, and two distinct Si-HAp-LEG formulations differing in gadolinium(III) (Gd3+) as well as lithium (Li+) and europium(III) (Eu3+) ion concentrations: Si-HAp-LEG-221 (1 mol% Gd3+ ion) and Si-HAp-LEG-222 (2 mol% Gd3+ ion). Differentiation-associated phenotypic outcomes, including extracellular matrix formation and lipid accumulation, were evaluated using Safranin O, Alizarin Red, and Oil Red O staining. In parallel, biomaterial-induced molecular responses were characterized at the transcriptomic and protein levels using RT-qPCR for selected coding and non-coding RNAs and Western blot analysis for representative lineage-associated proteins. Results: Histochemical evaluation confirmed that, across all tested biomaterial groups, BMSCs retained the ability to form mineralized calcium deposits, proteoglycan-rich extracellular matrix, and intracellular lipid accumulation under osteogenic, chondrogenic, and adipogenic conditions, respectively. Quantitative staining analysis revealed no significant Si-HAp-LEG-dependent enhancement of terminal differentiation outcomes compared with undoped Si-HAp. In turn, the molecular response differed between biomaterials modifications. Si-HAp-LEG-222 induced the most prominent changes in transcriptional and post-transcriptional regulators, particularly within BMP/SMAD-associated pathways under osteogenic and chondrogenic conditions, underlying a potential link between gadolinium concentration and osteogenic lineage commitment. However, these transcriptomic responses were not mirrored by consistent changes at the protein level. The results suggest that silicate-phosphate substituted hydroxyapatite co-doped with Li+, Eu3+, and Gd3+ ions primarily affects the early regulatory pathways associated with BMSCs differentiation rather than enhancing their terminal maturation. Discussion: In conclusion, the collective data indicate that Li+, Eu3+, and Gd3+ ions LEG co-doping broadens the multifunctional potential of Si-HAp by introducing imaging-related properties while preserving its underlying pro-regenerative character. Li+, Eu3+, and Gd3+ ions co-doped LEG-substituted Si-HAp may therefore be considered a compatible biomaterial platform that maintains BMSC cellular plasticity and supports balanced, differentiation-dependent modulation of lineage-associated molecular responses.

One crucial step during early osteogenesis is the embedding of osteoblasts within a collagen-rich extracellular matrix (osteoid), where they subsequently differentiate into a functional network of osteocytes. However, reconstructing 3D osteocyte networks in vitro remains a major challenge. We recently developed a synthetic microporous hydrogel to support the in vitro culture of 3D bone cell networks. Although matrix biodegradability facilitates cell-material interactions, the influence of micropores on bone tissue morphogenesis and differentiation remains poorly understood. Here, we investigate the effect of cell-scale micropores on bone cell morphogenesis and osteogenic differentiation in vitro. By exploiting polymerization-induced phase separation (PIPS) between 4-arm polyethylene glycol vinyl sulfones and dextran in the presence of hyaluronan, we generated matrix metalloproteinase-sensitive hydrogels with cell-scale micropores. Increasing the dextran concentration enlarged the average pore size from 4 m to 8 m, accompanied by a slight decrease in mechanical stiffness. Following encapsulation within these hydrogels, primary human osteoblasts remained highly viable. Hydrogels with larger pores supported extensive 3D cell network formation, whereas hydrogels with smaller pores exhibited enhanced osteogenic differentiation following 21 days of osteogenic culture. Together, these findings highlight that bone cells are sensitive to microporous physical cues and even minor changes over pore sizes can make an impact on osteocyte-like morphogenesis and differentiation in vitro.