Bioactive Materials

The repair of bone defects is faced with the dual challenges of limited autograft donors and single function of artificial materials. The development of new materials with mechanical adaptability, drug-controlled release and osteogenic induction has become a research hotspot in bone tissue engineering. Here, we developed an injectable photocrosslinkable hydrogel by integrating icariin (ICA)-loaded mesoporous silica nanoparticles (MSN) into gelatin methacryloyl (GelMA). The amino-functionalized MSN enhanced compressive modulus by 1.5-fold (p < 0.05) and enabled pH-responsive ICA release (91.2{+/-}4.2% cumulative release over 15 days). Our in vitro findings revealed the composite hydrogel promoted BMSC proliferation was 1.43{+/-}0.04 times that of GelMA group. And express excellent osteogenic differentiation ability (ALP activity: 3.25-fold; Mineralization: 5.01-fold). In vivo study, Micro-CT revealed significantly higher bone volume fraction (BV/TV) in rat calvarial defects at 12 weeks, with histology confirming mature trabecular bone formation. This MSN-mediated spatiotemporal delivery system synchronizes immunomodulation and osteogenesis, offering a promising strategy for non-load-bearing osseointegration.

Silk fibroin (SF) and sericin (SS), the two major proteins of silk, are attractive biomaterials that show great potential in regenerative medicine. However, their biochemical interactions with stem cells were not fully understood. Here, we employed multiomics to obtain a global view of the triggered cellular processes and pathways of MSCs by SF and SS. Integrated RNA-seq and proteomics revealed that SF and SS strongly enhanced the paracrine activity of MSCs through differentially activating integrin and glycolytic pathways, rather than directly regulating stem cell fate to initiate multiple but distinct biological processes in MSCs. Those specific paracrine signals of MSCs stimulated by SF and SS effectively promoted skin wound healing by influencing the behaviors of multiple resident cells in skin wound microenvironments. This study provides comprehensive and reliable insights into the cellular interactions with SF and SS, enabling future development of silk-based therapeutics for tissue engineering and stem cell therapy.

Impaired skin healing and progression into chronic wounds is a prevalent and growing medical problem. Porous, resorbable biomaterials can be used as temporary substrates placed into skin defects to support cell infiltration, neo-tissue formation, and remodeling of nonhealing wounds. Naturally-derived biomaterials have promising healing benefits, but their low mechanical properties and exuberant costs limit their performance and use. Synthetic materials can be affordably manufactured and tuned across a broader range of physiochemical properties, but opportunities remain for tailoring them for ideal host immune and regenerative responses. Polyesters are the most clinically-tested class of synthetic biomaterials, but their hydrolysis releases acidic degradation products that can cause autocatalytic degradation processes that are poorly controlled and are not tied to cellular or other biologic activities. Here, we systemically explored a series of ROS-degradable polythioketal (PTK) urethane (UR) foams with varied hydrophilicity as an alternative class of synthetic biomaterials for wound healing. It was found that the most hydrophilic PTK- UR variant, which had 7 ethylene glycol (EG7) repeats flanking each side of each thioketal bond, had the highest ROS reactivity of the PTK-URs tested. In an in vivo porcine excisional skin wound healing model, hydrophilic EG7 PTK-UR foams more effectively promoted tissue integration, ECM deposition, and re- epithelialization of full-thickness skin wound compared to more hydrophobic PTK-UR variants. Resolution of type 1 inflammation and lower foreign body response to scaffold remnants was also observed for EG7 versus more hydrophobic PTK-UR scaffolds. Finally, porcine wound healing studies showed that EG7 PTK-UR foams had similar wound healing response to a collagen-based clinical gold standard product, Integra Bilayer Wound Matrix (BWM), while outperforming polyester UR foam-based NovoSorb Biodegradable Temporizing Matrix (BTM) with respect to increased ECM production, vascularization, and biomaterial-associated immune phenotype. In sum, PTK-UR foams warrant further development toward a new class of synthetic biomaterial foams for skin wound healing applications.

Formation of chondromimetic human mesenchymal stem cells (hMSCs) condensations typically required in vitro culture in defined environments. In addition, extended in vitro culture in differentiation media over several weeks is usually necessary prior to implantation, which is costly, time consuming and delays clinical treatment. Here, this study reports on immediately implantable core/shell microgels with a high-density hMSC-laden core and rapidly degradable hydrogel shell. The hMSCs in the core formed cell condensates within 12 hours and the oxidized and methacrylated alginate (OMA) hydrogel shells were completely degraded within 3 days, enabling spontaneous and precipitous fusion of adjacent condensed aggregates. By delivering transforming growth factor-{beta}1 (TGF-{beta}1) within the core, the fused condensates were chondrogenically differentiated and formed cartilage microtissues. Importantly, these hMSC-laden core/shell microgels, fabricated without any in vitro culture, were subcutaneously implanted into mice and shown to form cartilage tissue via cellular condensations in the core after 3 weeks. This innovative approach to form cell condensations in situ without in vitro culture that can fuse together with each other and with host tissue and be matured into new tissue with incorporated bioactive signals, allows for immediate implantation and may be a platform strategy for cartilage regeneration and other tissue engineering applications.

This study aimed to develop a BMSC-laden polyethylene glycol diacrylate/methacrylated hyaluronic acid (PEGDA/HAMA) dual-crosslinked hydrogel and evaluate its effects on osteochondral defect repair. Two PEGDA concentrations, 3.75% and 7.5% (w/v), were used to prepare representative soft and stiff hydrogel formulations, respectively. The hydrogels were characterized in terms of morphology, cytocompatibility, and compressive behavior, and their ability to support BMSC-associated matrix deposition was evaluated in vitro. Repair outcomes were further assessed in a rat osteochondral defect model at 4 and 8 weeks. The 7.5% PEGDA/HAMA hydrogel exhibited higher stiffness than the 3.75% formulation and supported BMSC viability and matrix deposition in vitro. In vivo, the stiff hydrogel group showed improved defect filling and subchondral bone remodeling compared with the soft hydrogel and defect groups. However, histological and immunohistochemical analyses revealed predominant collagen type I deposition and limited collagen type II expression in the repair region, indicating fibrocartilaginous rather than hyaline-like cartilage repair. These findings suggest that BMSC-laden PEGDA/HAMA hydrogels may provide a useful platform for osteochondral defect repair, while further optimization of degradation behavior, matrix maturation, and collagen type II deposition is required to improve hyaline cartilage-oriented repair.

MicroRNA (miR)-200c suppresses the initiation and progression of oral squamous cell carcinoma (OSCC), the most prevalent head and neck cancer with high recurrence, metastasis, and mortality rates. However, miR-200c-based gene therapy to inhibit OSCC growth and metastasis has yet to be reported. To develop an miR-based gene therapy to improve the outcomes of OSCC treatment, this study investigates the feasibility of plasmid DNA encoding miR-200c delivered via non-viral CaCO3-based nanoparticles to inhibit OSCC tumor growth. CaCO3-based nanoparticles with various ratios of CaCO3 and protamine sulfate (PS) were utilized to transfect pDNA encoding miR-200c into OSCC cells and the efficiency of these nanoparticles was evaluated. The proliferation, migration, and associated oncogene production, as well as in vivo tumor growth for OSCC cells overexpressing miR-200c were also quantified. It was observed that, while CaCO3-based nanoparticles improve transfection efficiencies of pDNA miR-200c, the ratio of CaCO3 to PS significantly influences the transfection efficiency. Overexpression of miR-200c significantly reduced proliferation, migration, and oncogene expression of OSCC cells, as well as the tumor size of cell line-derived xenografts (CDX) in mice. In addition, a local administration of pDNA miR-200c using CaCO3 delivery significantly enhanced miR-200c transfection and suppressed tumor growth of CDX in mice. These results strongly indicate that the nanocomplexes of CaCO3/pDNA miR-200c may potentially be used to reduce oral cancer recurrence and metastasis and improve clinical outcomes in OSCC treatment. (227 words)

Traumatic cranial defects often involve concurrent loss of soft and hard tissues and can progress to chronic defects due to delayed healing associated with infection or other co-morbidities. Despite autologous reconstruction remaining the clinical standard, it requires staged procedures using heterogeneous tissues, increasing operative time, costs, and surgical risks. Moreover, current tissue engineering approaches focus on single tissues or acute tissue defect models, limiting their clinical applications. Herein, we describe an acellular, material-driven 3D-printed composite scaffold designed to regenerate both bone and skin within composite cranial defects. The scaffold integrates controlled copper ion release from both organic and inorganic components with 3D-printed citrate polymer and citrate polymer-ceramic composites. Integrated thermoresponsive citrate-based hydrogels further enable spatially defined dermoconductive and osteoconductive properties, supporting a one-step surgical approach. At 12 weeks post-implantation, our scaffold enhanced keratinocyte organization, collagen deposition, and defect coverage with mature bone, achieving histological outcomes comparable to autografts. Furthermore, the system suppressed bacterial burden. Thus, this acellular platform represents a clinically promising synchronized strategy to address the complex demands of traumatic craniofacial composite defects.

Cancer models in animal studies play a central role in cancer research, particularly in investigating vascularized tumor tissues for the validation of immune cell therapies. However, xenografts relying solely on cancer cells are ineffective for optimal tumor tissue formation. Additionally, tumor modeling using hydrogels with cancer cells to promote vascularization often leaves behind residual biomaterials that inhibit integration with surrounding tissues. To address these issues, we utilized a straightforward in vivo vascularized tumor modeling method with a completely degradable, crosslinker-free carboxymethyl chitosan (CMCTS)/oxidized hyaluronic acid (OHA) hydrogel that encapsulates high-density human cancer cells for in situ injection. The CMCTS/oHA hydrogel was fully degraded within 3 weeks, enabling three-dimensional (3D) cell condensation in vitro. 2 weeks after subcutaneous injection in mice, solid tumors formed, with native host vasculature infiltrating the transplanted human cancer cells, confirming spontaneous hydrogel degradation. Following this, human macrophages were administered via tail vein injection, enhancing the accumulation of mouse immune cells in the humanized tumor twofold and showing murine macrophages adjacent to the vasculature. This study thus provides proof-of-concept for a facile and fully vascularized humanized tumor model in mice for validating immune cell therapies. HIGHLIGHTSO_LIThe oHA was prepared using sodium periodate treatment, which facilitated the formation of in situ CMCTS/oHA hydrogels C_LIO_LICMCTS/oHA hydrogels completely degraded within a short period, allowing for 3D cell condensation C_LIO_LIHigh-density cell-laden CMCTS/oHA hydrogels were injected subcutaneously in mice, resulting in the generation of a vascularized solid tumor C_LIO_LIThe transplanted therapeutic cell was observed to adhere to the tumor tissue through the bloodstream C_LI

We produced a three dimensional (3D) bone tumor model (BTM) to study the interactions between healthy and tumor cells in a tumor tissue microenvironment, migration of the tumor cells and the efficacy of an anticancer drug, Doxorubicin, for personalized medicine applications. The model consisted of two compartments: (a) a healthy bone tissue mimic, poly(lactic acid-co-glycolic acid) (PLGA)/beta-tricalcium phosphate ({beta}-TCP) sponge that was seeded with human fetal osteoblastic cells (hFOB) and human umbilical vein endothelial cells (HUVECs), and (b) a tumor mimic, a lyophilized collagen sponge that was seeded with human osteosarcoma cells (Saos-2). The tumor component was introduced to a central cavity created in the healthy bone mimic and together they constituted the total 3D model (3D-BTM). The scaffolds were characterized by determining their mechanical properties, studying their topography and stability with compression tests, microCT, SEM, confocal microscopy and gravimetry. Porosities of the sponges were determined from {micro}CT data as 96.7% and 86% for PLGA/TCP and collagen sponges, respectively. The average diameters of the pores were measured by using ImageJ (NIH, USA) as 199{+/-}52 {micro}m for PLGA/TCP and 50-150 m for collagen scaffolds. Young modulus of the PLGA/TCP and collagen sponges were determined as 4.76MPa and 140kPa, respectively. Cells seeded on the two sponges were studied independently and together on the BTM. Cell proliferation, morphology, calcium phosphate forming capacity and ALP production were studied on both healthy bone and tumor mimics. All types of cells showed cellular extensions and spread on and in the scaffolds indicating good cell-material interactions. Angiogenic developments in BTM were studied along with migration of cells between the components with immunocytochemistry, SEM, microCT, qRT-PCR and agarose gel electrophoresis. Confocal microscopy showed that a direct contact was established between the cells present in different parts of the BTM; and the HUVEC cells within the healthy bone mimic were observed to migrate into the tumor mimic. This was confirmed by the increase in the levels of angiogenic factors VEGF, bFGF, and IL-8 in the tumor component. The IC50 of Doxorubicin on Saos-2 cells was determined as 0.1876 {micro}g.mL-1. Doxorubicin was administered to the BTM at 2.7 {micro}g.mL-1 concentration and after allowing one day for interaction, the cell number was determined with Alamar Blue cell viability test as 7-fold lesser compared to 24 h earlier. Apoptosis of the osteosarcoma cells was measured by caspase-3 enzyme activity assay. These results demonstrate the suitability of the 3D BTM model for use in the investigation of activities and migrations of cells in a tumor tissue. These will be very useful in studying metastatic capabilities of cells in addition to personalized drug treatments.

Bone tissue engineering involves the usage of metals, polymers, and ceramics as the base constituents in the fabrication of various biomaterial 3D scaffolds. Of late, the composite materials facilitating enhanced osteogenic differentiation/regeneration have been endorsed as the ideally suited bone grafts for addressing critical-sized bone defects. Here, we report the successful fabrication of 3D composite scaffolds with collagen type I (Col-I) in conjunction with three different crystalline phases of calcium-phosphate (CP) nanomaterials [hydroxyapatite (HAp), beta-tricalcium phosphate ({beta}TCP), biphasic hydroxyapatite ({beta}TCP-HAp or BCP)], obtained by altering the pH as the major variable. The fabricated 3D scaffolds consisting of [~]70 wt % CP nanomaterials and [~] 30 Wt % of Col-I did mimic the ECM of bone tissue. The different Ca/P ratio and the orientation of CP nanomaterials in CP/Col-I composite scaffolds altered the microstructure, surface area, porosity, and mechanical strength of the scaffolds and also influenced the bioactivity, biocompatibility, and osteogenic differentiation of gingival-derived mesenchymal stem cells (gMSCs). The microstructure of CP/Col-I 3D scaffolds assessed by Micro-CT analysis revealed randomly oriented interconnected pores with pore sizes ranging from 80-250, 125-380, and 100-450{micro}m respectively for {beta}TCP/Col-I, BCP/Col-I, and HAp/Col-I scaffolds. Among these, the BCP/Col-I achieved the highest surface area ([~] 42.6 m2/g) and porosity ([~]85%), demonstrated improved bioactivity and biocompatibility, and promoted osteogenic differentiation of gMSCs. Interestingly, the Ca2+ ions (3 mM) released from scaffolds could also facilitate the osteocyte differentiation of gMSCs sans osteoinduction. Collectively, our study has demonstrated the ECM mimicking biphasic CP/Col-I 3D scaffold as an ideally suited tissue-engineered bone graft.

Orofacial clefts are the most common craniofacial congenital anomaly. Following cleft palate repair, up to 60% of surgeries have wound healing complications leading to oronasal fistula (ONF), a persistent connection between the roof of the mouth and the nasal cavity. The current gold standard methods for ONF repair use human allograft tissues; however, these procedures have risks of graft infection and/or rejection, requiring surgical revisions. Immunoregenerative therapies present a novel alternative approach to harness the bodys immune response and enhance the wound healing environment. We utilized a repurposed FDA-approved immunomodulatory drug, FTY720, to reduce the egress of lymphocytes and induce immune cell fate switching toward pro-regenerative phenotypes. Here, we engineered a bilayer biomaterial system using Tegaderm, a liquid-impermeable wound dressing, to secure and control the delivery of FTY720- nanofiber scaffolds (FTY720-NF). We optimized release kinetics of the bilayer FTY720-NF to sustain drug release for up to 7d with safe, efficacious transdermal absorption and tissue biodistribution. Through comprehensive immunophenotyping, our results illustrate a pseudotime pro-regenerative state transition in recruited hybrid immune cells to the wound site. Additional histological assessments established a significant difference in full thickness ONF closure in mice on Day 7 following treatment with bilayer FTY720-NF, compared to controls. These findings demonstrate the utility of immunomodulatory strategies for oral wound healing, better positing the field to develop more efficacious treatment options for pediatric patients. One Sentence SummaryLocal delivery of bilayer FTY720-nanofiber scaffolds in an ONF mouse model promotes complete wound closure through modulation of pro-regenerative immune and stromal cells.

Controlled delivery of growth factors and viable cells remains a significant challenge in bone tissue engineering. In this study, a 3D-printed hydrogel scaffold system was developed for the co-delivery of bone morphogenetic protein-9 (BMP-9) and preosteoblasts to enhance bone regeneration. The system consisted of a 3D-printed base scaffold containing BMP-9-coated calcium sulfate (CaS) microparticles and a photocurable hydrogel coating layer encapsulating viable cells. The scaffold design exploited electrostatic interactions between BMP-9 and gelatin matrices by incorporating gelatin type B in the base scaffold and gelatin type A in the coating layer. Differences in the isoelectric points of these gelatin types were utilized to regulate protein binding and release. Release studies demonstrated that CaS microparticles alone exhibited rapid burst release, with nearly 80% of BMP-9 released within 24 h. Encapsulation of BMP-9 coated CaS particles in the 3D-printed scaffolds reduced the release rate, while the addition of the coating layer significantly improved sustained release, limiting BMP-9 release to approximately 50-60% by day 5. Bioactivity studies showed enhanced cell attachment in BMP-9 containing scaffolds compared with controls. Live/Dead cytotoxicity assays demonstrated high cell viability (>80%) within the coating layer over the culture period, confirming that the encapsulation and photocuring processes did not adversely affect cell survival. Cell proliferation and differentiation were further evaluated using WST-1 and alkaline phosphatase assays. The results demonstrate that electrostatic interactions governed by gelatin type selection can regulate BMP-9 release while maintaining high cell viability, providing a promising platform for growth factors and cell delivery in bone tissue engineering.

Reconstruction of bone defect is one of the difficult problems in orthopedic treatment, and bone tissue scaffold implantation is the most promising direction of bone defect reconstruction. In this study, we used the combination of HA (Hydroxyapatite) and PLGA [Poly (lactic-co-glycolic acid)] in the construction of polymer scaffolds, and introduced bioactive MSM (Methyl sulfonyl methane) into polymer scaffolds to prepare porous scaffolds. The osteoblasts, isolated and cultured in vitro, were seeded in the porous scaffolds to construct tissue-engineered scaffolds. Meanwhile, the model of rabbit radius defect was constructed to evaluate the biological aspects of five tissue-engineered scaffolds, which provided experimental basis for the application of the porous scaffolds in bone tissue engineering. The SEM characterization showed the pore size of porous scaffolds was uniform and the porosity was about 90%. The results of contact Angle testing suggested that the hydrophobic porous scaffold surface could effectively promote cell adhesion and cell proliferation, while mechanical property test showed good machinability. The results of drug loading and release efficiency of MSM showed that porous scaffolds could load MSM efficiently and prolong the release time of MSM. In vitro incubation of porous scaffolds and osteoblasts showed that the addition of a small quantity of MSM could promote the infiltration and proliferation of osteoblasts on the porous scaffolds. Similar results were obtained by implanting the tissue-engineered scaffolds, fused with the osteoblasts and MSM/HA/PLGA porous scaffolds, into the rabbit radius defect, which provided experimental basis for the application of the MSM/HA/PLGA porous scaffolds in bone tissue engineering.

Wound healing is a complex biological process critical for restoring skin integrity after injury. However, chronic wounds present significant clinical challenges due to persistent inflammation, disrupted collagen synthesis, and susceptibility to infection. Bioactive scaffolds have emerged as promising therapeutic strategies to enhance tissue regeneration by modulating cellular behavior and extracellular matrix (ECM) dynamics. This study explores a hyaluronic acid-collagen (HyCol) scaffold enriched with vitamin C (VC), producing (VC-HyCol) to improve wound healing in preclinical rat models. Hyaluronic acid and collagen, key ECM components, provide structural and biochemical support, while vitamin C acts as both a collagen biosynthesis cofactor and an antioxidant to counteract oxidative stress. The scaffold was designed to emulate the native ECM microenvironment, facilitating fibroblast proliferation, keratinocyte migration, and angiogenesis. Physicochemical characterization, biocompatibility assessments, and in vivo wound healing experiments were performed to evaluate its therapeutic efficacy. Results demonstrated that the incorporation of vitamin C significantly enhanced fibroblast activity, reduced inflammatory markers, and accelerated tissue regeneration compared to control groups. Histological and molecular analyses further confirmed enhanced collagen deposition and neovascularization, indicating faster and more organized wound repair. These findings highlight the potential of this multifunctional scaffold as an advanced wound dressing, with significant implications for regenerative medicine and clinical wound management. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/665568v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@d8d248org.highwire.dtl.DTLVardef@d58e05org.highwire.dtl.DTLVardef@5f210eorg.highwire.dtl.DTLVardef@17354b1_HPS_FORMAT_FIGEXP M_FIG C_FIG

Chronic wound healing is often impaired in conditions such as metabolic syndrome, requiring effective therapeutic interventions to promote tissue regeneration and repair. In this study, we evaluated the wound healing potential of petroleum jelly (P Jelly)-based bioactive glass ointments (PBGCu) with varying copper concentrations (0, 1, and 3 wt%) in both in vitro and in vivo models of wound healing. PBGCu formulations demonstrated high biocompatibility with human dermal fibroblasts (HDF) and human umbilical vein endothelial cells (HUVEC). Additionally, PBGCu ointments exhibited strong antibacterial activity against Staphylococcus aureus, suggesting their utility for the care of chronic wounds. In both metabolic syndrome mouse and pig models, PBGCu3-treated wounds showed significantly faster wound closure, enhanced epithelial regeneration, and increased dermal thickness compared to saline and P Jelly controls. Histological analysis also revealed 50% increased vascularization (p < 0.0001) and a 90% reduction in scar formation (p < 0.0001) in PBGCu3-treated wounds. These findings show that PBGCu formulations, especially at 3 wt% copper concentration, significantly improve wound healing by promoting epithelial regeneration, dermal tissue formation, and vascularization, while also offering antibacterial protection. The sustained Cu2+ ions release from PBGCu ointments provides long-term support for tissue regeneration, positioning this ointment composition as a promising therapeutic tool for chronic wound management. Future studies will focus on elucidating the underlying mechanisms and evaluating the therapeutic efficacy of PBGCu formulations in infected wounds. HighlightsO_LIDeveloped a Petroleum Jelly-based copper-doped bioactive glass ointment (PBGCu) enabling sustained and controlled Cu{superscript 2} ion release. C_LIO_LIPBGCu significantly accelerated wound closure and improved epithelial and dermal tissue regeneration. C_LIO_LIPBGCu enhanced hair follicle regeneration and tissue remodeling in full-thickness wounds. C_LIO_LIValidated therapeutic efficacy in both mouse and pig models that support translational relevance. C_LIO_LIOffers a simple, low-cost, and clinically adaptable topical formulation for metabolic syndrome-related wound complications. C_LI

Periodontitis is an inflammatory infection caused by bacterial plaque accumulation that affects the periodontium, a complex structure of different tissues (cementum, periodontal ligament and alveolar bone) that surrounds and supports the teeth. Current treatments lack bioactive signals to induce tissue repair and coordinated regeneration of the periodontium, thus alternative strategies are needed to improve clinical outcomes. Cell-derived extracellular matrix (ECM) has been combined with biomaterials to enhance their biofunctionality for various tissue engineering (TE) applications. In this work, bioactive cell-derived ECM loaded electrospun polycaprolactone/chitosan (PCL/CTS) nanofibrous scaffolds were developed combining polymer solutions with lyophilized decellularized ECM (dECM) derived from human Periodontal Ligament Stem/Stromal Cells (PDLSCs). The works aims were to fabricate and characterize cell-derived ECM electrospun PCL/CTS scaffolds in terms of morphology, physico-chemical, thermal and mechanical properties and assess their ability to enhance the osteogenic differentiation of PDLSCs, envisaging periodontal TE applications. PDLSCs were cultured and used for dECM production. PDLSCs-derived dECM was characterized regarding morphology, protein expression, DNA removal efficiency, and glycosaminoglycans and collagen contents. Osteogenic differentiation of PDLSCs was performed on PCL, PCL/CTS and PCL/CTS/ECM electrospun scaffolds for 21 days. The obtained results demonstrate that PCL/CTS/ECM scaffolds promoted cell proliferation compared to PCL and PCL/CTS scaffolds, while maintaining similar physical and mechanical properties of PCL/CTS scaffolds. PCL/CTS/ECM scaffolds enhanced the osteogenic differentiation of PDLSCs, confirmed by increased alkaline phosphatase activity, calcium deposition, and bone-specific marker genes expression. Moreover, PCL/CTS scaffolds showed higher levels of cell mineralization than PCL scaffolds. Overall, this work describes the first use of lyophilized cell-derived ECM loaded electrospun scaffolds for periodontal TE applications and highlights its potential as a promising therapeutic strategy for periodontitis treatment.

Collagens, the predominant structural proteins in mammalian, maintain tissue integrity through its unique triple-helical conformation. Traditionally, cosmetic collagens have been administered as hydrolyzed peptides derived from animal sources, which exhibit poor bioavailability and functional efficacy due to limited skin penetration and disrupted tertiary/quaternary structures. To overcome these limitations, we developed a novel system for in vivo collagen expression. This system utilizes the topical application of in vitro synthesized, AI-designed mRNAs encoding types I, III, and XVII collagens to improve human skin condition. Significant upregulation of target collagen expression was observed in dermal fibroblasts, leading to improved skin condition through in situ assembly of collagens in natural conformation.

Mesenchymal stem cell (MSC) transplantation was suggested as a promising approach to treat spinal cord injury (SCI). However, the heterogeneity of MSC and the lack of appropriate delivery methods impede its clinical application. To tackle these challenges, we first generated human MSCs derived from a single cell with a great homogeneity of batch quality and then developed a biocompatible injectable hydrogel to embed these cells to treat severe SCI. In a clinically relevant rat severe SCI model, we showed that the injection of MSCs with injectable hydrogel into the lesion site promoted robust functional recovery, while the intrathecal delivery of MSCs only resulted in limited therapeutic effects. Mechanistically, the hydrogel protected MSCs from the damage of harmful neuroinflammatory microenvironment in the spinal cord lesion. The hydrogel with the survived MSCs ameliorates the neuroinflammatory microenvironment of spinal cord lesion, preventing cavity formation and leads to the remnant of spared axons/tissues, which results in a better prognosis in the end.

Burn wounds are a common traumatic injury that impair cellular function and hinder the healing process, often resulting in significant skin loss. While autologous skin grafting is considered the gold standard for treating burns, its widespread use is limited due to donor site morbidity and the requirement for large amounts of tissue. Traditional wound dressings and treatments often fail to ensure complete recovery. Being initially FDA-approved to treat multiple sclerosis, 4-aminopyridine (4-AP) has also been shown to accelerate burn wound closure by transforming keratinocytes and fibroblasts when administered systemically. However, prolonged systemic use of 4-AP can lead to significant side effects. In this study, we aimed to repurpose 4-AP for treating skin burn wounds by delivering it topically using a laponite-gelatin gel formulation. This method allows for non-invasive and localized drug delivery on burn wound site. We evaluated the physical properties of the 4-AP gel shear thinning behavior, drug release kinetics, biocompatibility, and functional wound closure using a scratch assay. Moreover, our in vivo experiments showed that the 4-AP loaded gel accelerates wound healing by enhancing re-epithelialization and hair follicle regeneration and promoting fibroblast to myofibroblast transformation, which supports extracellular matrix remodeling after skin burns. This novel application of the 4-AP gel could offer a promising alternative to current burn wound therapies, potentially leading to improved outcomes for burn patients.

Small non-coding RNAs (sncRNA) hold promising therapeutic potential. However, their clinical application is hindered by the poor cytocompatibility and limited transfection efficiency of conventional delivery vectors. In contrast, red blood cell-derived extracellular vesicles (RBCEVs) offer a safer, more efficient, and cost-effective alternative. Given the limited studies on the application of RBCEVs in the central nervous system (CNS) which is characterized by the presence of sensitive cell types with inherently low transfection efficiency, we hypothesized that RBCEVs could serve as a safe and effective sncRNA delivery vector for CNS applications, and that their incorporation into 3D-printed scaffolds could enable sustained and localized delivery of therapeutic sncRNAs. To test this, the uptake and gene silencing performance of RBCEVs were examined in primary CNS cell types, including astrocytes, neurons, oligodendrocyte precursor cells (OPCs), and microglia. While over 70% of OPCs and microglia internalized RBCEVs, uptake in neurons and astrocytes remained below 40%, indicating cell-type-specific uptake efficiency. Additionally, RBCEVs-mediated delivery of siRNA resulted in the highest gene knockdown efficiency in OPCs (74.2%), while triggering less than 30% gene knockdown in other cell types. Next, RBCEVs-encapsulated scaffolds were fabricated using digital light processing (DLP) 3D printing, enabling the sustained release of miR-219/miR-338-loaded RBCEVs for at least 21 days in vitro, which resulted in effective gene silencing that promoted OPC differentiation and myelination. Using spinal cord injury (SCI) as a proof-of-concept, scaffold-mediated delivery of RBCEVs-miR-219/miR-338 significantly promoted OPC differentiation and maturation in vivo as evidenced by increased CC1 mature oligodendrocytes and reduced PDGFR undifferentiated OPCs (p < 0.001). Taken together, these results demonstrate the therapeutic potential of combining RBCEVs with DLP-printed scaffolds for localized and sustained sncRNA delivery in CNS disease treatment.