Bioactive Materials

A large population of people is affected by obesity (OB) and its associated type 2 diabetes mellitus(T2DM). There are currently no safe and long-lasting anti-OB/T2DM therapies. Clinical data and preclinical transplantation studies show that transplanting metabolically active brown adipose tissue (BAT) is a promising approach to prevent and treat OB and its associated metabolic and cardiovascular diseases. However, most transplantation studies used mouse BAT, and it is uncertain whether the therapeutic effect would be applied to human BAT since human and mouse BATs have distinct differences. Here, we report the fabrication of three-dimensional (3D) human brown adipose microtissues, their survival and safety, and their capability to improve glucose and insulin homeostasis and manage body weight gain in high-fat diet (HFD)-induced OB and diabetic mice. Methods3D BA microtissues were fabricated and transplanted into the kidney capsule of Rag1-/- mice. HFD was initiated to induce OB 18 days after transplantation. A low dose of streptozotocin (STZ) was administrated after three months HFD to induce diabetes. The body weight, fat and lean mass, plasma glucose level, glucose tolerance and insulin sensitivity were recorded regularly. In addition, the levels of human and mouse adipokines in the serum were measured, and various tissues were harvested for histological and immunostaining analyses. ResultsWe showed that 3D culture promoted BA differentiation and uncoupling protein-1 (UCP-1) protein expression, and the microtissue size significantly influenced the differentiation efficiency and UCP-1 protein level. The optimal microtissue diameter was about 100 {micro}m. Engineered 3D BA microtissues survived for the long term with angiogenesis and innervation, alleviated body weight and fat gain, and significantly improved glucose tolerance and insulin sensitivity. They protected the endogenous BAT from whitening and reduced mouse white adipose tissue (WAT) hypertrophy and liver steatosis. In addition, the microtissues secreted soluble factors and modulated the expression of mouse adipokines. We also showed that scaling up the microtissue production could be achieved using the 3D suspension culture or a 3D thermoreversible hydrogel matrix. Further, these microtissues can be preserved at room temperature for 24 hours or be cryopreserved for the long term without significantly sacrificing cell viability. ConclusionOur study showed that 3D BA microtissues could be fabricated at large scales, cryopreserved for the long term, and delivered via injection. BAs in the microtissues had higher purity, and higher UCP-1 protein expression than BAs prepared via 2D culture. In addition, 3D BA microtissues had good in vivo survival and tissue integration, and had no uncontrolled tissue overgrowth. Furthermore, they showed good efficacy in preventing OB and T2DM with a very low dosage compared to literature studies. Thus, our results show engineered 3D BA microtissues are promising anti-OB/T2DM therapeutics. They have considerable advantages over dissociated BAs or BAPs for future clinical applications in terms of product scalability, storage, purity, quality, and in vivo safety, dosage, survival, integration, and efficacy.

Mesenchymal stem cells (MSCs) offer significant therapeutic potential, but traditional 2D culture on rigid substrates results in progressive cell enlargement and senescence, reducing proliferative capacity and therapeutic potency. This poses a major challenge for widespread clinical application. We explored a novel strategy for placenta-derived MSCs combining 2D expansion with 3D spheroid culture to address these limitations. Our research shows that culturing MSCs as 3D spheroids significantly reduces individual cell size and size distribution compared to 2D culture. Spheroid formation is feasible in chemically defined media with minimal cell death, and is enhanced by extracellular matrix protein supplementation. Although MSCs do not proliferate in 3D suspension, an alternating 2D/3D culture protocol, transitioning cells between 2D flasks and 3D spheroids after each passage, effectively slows MSC enlargement and senescence over long periods. This alternating method also preserves MSC immunomodulatory function, unlike continuous 2D culture which leads to its loss. For scalability, we developed an RGD-functionalized alginate hydrogel tube (AlgTube) system that mimics this alternating environment, supporting both adherent growth and chemically triggered spheroid formation. This alternating 2D/3D culture strategy and the scalable AlgTube platform provide a foundation for developing next-generation MSC manufacturing technologies to meet future clinical demands.

Bone defects may occur in different sizes and shapes due to trauma, infections, and cancer resection. Autografts are still considered the primary treatment choice for bone regeneration. However, they are hard to source and often create donor-site morbidity. Injectable microgels have attracted much attention in tissue engineering and regenerative medicine due to their ability to replace inert implants with a minimally invasive delivery. Here, we developed novel cell-laden bioprinted gelatin methacrylate (GelMA) injectable microgels, with controllable shapes and sizes that can be controllably mineralized on the nanoscale, while stimulating the response of cells embedded within the matrix. The injectable microgels were mineralized using a calcium and phosphate-rich medium that resulted in nanoscale crystalline hydroxyapatite deposition and increased stiffness within the crosslinked matrix of bioprinted GelMA microparticles. Next, we studied the effect of mineralization in osteocytes, a key bone homeostasis regulator. Viability stains showed that osteocytes were maintained at 98% viability after mineralization with elevated expression of sclerostin in mineralized compared to non-mineralized microgels, indicating that mineralization effectively enhances osteocyte maturation. Based on our findings, bioprinted mineralized GelMA microgels appear to be an efficient material to approximate the bone microarchitecture and composition with desirable control of sample injectability and polymerization. These bone-like bioprinted mineralized biomaterials are exciting platforms for potential minimally invasive translational methods in bone regenerative therapies.

A novel bio-active scaffold for enhancing wounded meniscus healing has been developed by combination of High mobility group box 1 protein (HMGB1) and kartogenin (KGN) with alginate gel. The properties of the bio-active scaffold were also investigated using an in vitro cell culture model and in vivo rat wounded meniscus healing model. This HMGB1-KGN-containing bioactive scaffold released HMGB1 and KGN into wound area and kept high concentrations of HMGB1 and KGN in the system for more than two weeks. This HMGB1-KGN-containing bioactive scaffold also activated rat bone marrow stem cells (BMSCs) from G0 to GAlert stage and promoted cell proliferation as evidenced by 5-bromo-2-deoxyuridine (BrdU) incorporation testing. Our results also demonstrated that the HMGB1-KGN-containing bioactive scaffold induced cell migration in vitro and recruited the cells to wound area to promote wounded rat meniscus healing in vivo.

Mesenchymal stem cells (MSCs)-based bone tissue regeneration has gained significant attention due to their excellent differentiation capacity and immunomodulatory activity. Enhancing osteogenesis regulation is crucial for improving the therapeutic efficacy of MSC- based regeneration. By utilizing the regenerative capacity of bone ECM and the functionality of nanoparticles, we recently engineered bone-based nanoparticles (BNPs) from decellularized porcine bone. The effects of internalization of BNPs on MSCs viability, proliferation, and osteogenic differentiation were first investigated and compared at different time points. The phenotypic behaviors, including cell number, proliferation, and differentiation were characterized and compared. By incorporating this LNA/DNA nanobiosensor and MSCs live cell imaging, we monitored and compared Notch ligand delta-like 4 (Dll4) expression dynamics in cytoplasm and nucleus during osteogenic differentiation. Pharmacological interventions are used to inhibit Notch signaling to examine the mechanisms involved. The results suggest Notch inhibition mediates osteogenic process, with reduced expression of early and late stage of differentiation markers (ALP, calcium mineralization). The internalization of BNPs led to an increase in Dll4 expression, exhibiting a time-dependent pattern that aligned with enhanced cell proliferation and differentiation. Our findings indicate that the observed changes in BNP-treated cells during osteogenic differentiation could be associated with the elevated levels of Dll4 mRNA expression. In summary, this study provides new insights into MSCs osteogenic differentiation and the molecular mechanisms through which BNPs stimulate this process. The results indicate that BNPs influence osteogenesis by modulating Notch ligand Dll4 expression, demonstrating a potential link between Notch signaling and the proteins present in BNPs.

Chronic wounds present a significant clinical challenge due to impaired skin microvasculature, particularly in diabetes. The tissue engineering study introduces therapeutic Exo-Q, a unique thermoresponsive polymer hydrogel created with Q protein nanofibers and cultured human bone marrow multipotent stromal cell exosomes with consistent gene signature. Limited, local and topical application of Exo-Q hydrogel is feasible for maximum neovascularization during murine diabetic wound closure in a xenotransplantation model, as well as compatibility and vascular delivery in human skin in a xenograft model. Exo-Q hydrogel treatment significantly reduces diabetic wound closure time within ranges of non-diabetic wounds. This innovative, non-invasive tissue engineered therapeutic option offers a promising approach to addressing the complex pathologies of non-healing wounds.

Recently, microgels have been widely used in three-dimensional (3D) bioprinting as both supporting baths and bioinks. As a bioink, microgels have several unique properties, such as shear-thinning and self-healing behaviors with tunable mechanics, making them useful in 3D bioprinting. While cell encapsulated microgels offer many advantages in 3D bioprinting, they also have some limitations. It is still challenging to produce large quantities of cell encapsulated microgels with consistent quality and properties due to processes that are often complex and time-consuming. In this study, stem cell encapsulated, photocrosslinkable, shear-thinning and self-healing alginate microgel (SSAM) bioinks have been successfully fabricated via simple mixing of an oxidized and methacrylated alginate solution with suspended stem cells and a supersaturated calcium sulfate slurry solution through a custom-made spiral mixing unit. The SSAM bioinks can be bioprinted into complex 3D structures with both high resolution and shape fidelity due to their shear-thinning and self-healing properties. The 3D bioprinted SSAM bioinks can then serve as a supporting bath for the creation of prevascular network patterns using an individual cell-only prevasculogenic bioink within the 3D printed constructs. The prevascular network patterned 3D bioprinted constructs can be further stabilized by secondary photocrosslinking of the SSAMs, which enables long-term culture of the printed constructs for functional vascularized osteogenic tissue formation by differentiation of the bioprinted cells. The SSAM bioinks and individual cell-only printing technique enable in situ bioprinting of prevascularized tissue constructs in a mouse calvarial bone defect, achieving mechanical stability and ensuring the in situ bioprinted constructs remain within the defect.

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.

AbstractGrowth factors play a crucial role in regulating various cellular functions, including proliferation and differentiation. Consequently, the biomaterial-based delivery of exogenous growth factors presents a promising strategy in regenerative medicine to manage the healing process and restore tissue function. For effective therapeutic applications, it is essential that these active compounds are precisely targeted to the site of regeneration, with release kinetics that align with the slow pace of tissue growth. We have developed an ex vivo model utilizing a developing embryonic chick bone, and using PLGA based microparticles as controlled-release systems, allowing for the investigation of spatiotemporal effects of growth factor delivery on cell differentiation and tissue formation. Our findings demonstrate that BMP2 and FGF2 can significantly alter cell morphology and zonally pattern collagen deposition within the model, but only when the growth factor presentation rate is carefully regulated. Furthermore, the growth factor-dependent responses observed underscore the potential of this model to explore the interactions between cells and the growth factors released from biomaterials in an approach which can be applied for bone tissue engineering.

Traumatic dental tissue injury is a common health concern globally, with more than a billion people affected worldwide. In this study, we have synthesized green route nanohydroxyapatite utilizing green tea extract, which further enhances the osteogenesis, osteointegration, and biocompatibility of the synthesized hydroxyapatite. This Nanohydroxyapatite was capped with an antibiotic, integrated into an alginate scaffold, and coated with a painkiller to create a dual drug release system, providing rapid release of painkiller and sustained release of antibacterial drug. Biocompatibility and osteoblast proliferation were confirmed through MTT assays, and osteogenesis by ALP activity, validating the suitability of the scaffold for dental bone tissue engineering applications. The developed nanocomposite scaffold shows significant promise in enhancing dental bone repair by simultaneously addressing the inflammation, infection, and regeneration challenges. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=129 SRC="FIGDIR/small/693830v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@124b53forg.highwire.dtl.DTLVardef@f75868org.highwire.dtl.DTLVardef@cd20a6org.highwire.dtl.DTLVardef@11a7dfb_HPS_FORMAT_FIGEXP M_FIG C_FIG

Functional tissue regeneration in tissue engineering has long been an aspiration of humanity. However, conventional materials such as metal, ceramic, polymer, and decellularized extracellular matrix (dECM) have not yet yielded the desired outcomes. We have developed a collagen biomimetic material based on soluble collagen. This biomimetic material emulates the composition, structure, and function of native tissue. It possesses high strength, high affinity, high nutrient, and low antigenicity. The biomimetic material had been utilized to fabricate some implants (absorbable surgical suture, artificial tendon, hernia patch) and had demonstrated remarkable functional regeneration in animal experiments. In our previous researches, collagen surgical sutures facilitated wound healing, artificial tendons and artificial muscles (hernia patches) successfully repaired extensive defects of tendons and muscles, respectively. These successful tissue engineering endeavors enabled damaged defect to restore its biological functions and structure. Degradation of these biomimetic implants synchronized with the regeneration of the new tissues. Based on these successful functional regenerations, the present study focuses on regeneration of rabbit sciatic nerve. This collagen biomimetic material was employed to construct an artificial nerve tube (length: 2 cm, inner diameter: 2 mm, outer diameter: 3.5 mm) to repair a 2 cm-long defect in the rabbit sciatic nerve. After 36 weeks, the newly formed functional sciatic nerve successfully bridged the damaged ends, while approximately 92% of the artificial nerve tube has degraded. In this study, we provide an overview of the biomimetic material and its medical applications. We believe that these successful regenerations of tendon, muscle, and nerve all stem from the exceptional characteristics of this biomimetic material. We firmly believe that this collagen biomimetic material exhibits remarkable regenerative capabilities in diverse tissues. It can pave the way for the fabrication of increasingly critical artificial implants, which can be utilized to repair defects in human tissues, including artificial esophagi, tracheae, and small blood vessels. In regenerative medicine of induced pluripotent stem cells (iPS), the biomimetic materials can replace temperature-sensitive materials (PIPAAm) to fabricate cell sheets. This approach expedites the process, enhances cellular vitality, and consequently improves regeneration outcomes. This material technology not only provides a revolutionary solution for clinical treatment but also paves the way for a new development direction in regenerative medicine.

Mesenchymal stromal cells (MSC) are sensors of inflammation, and they exert immunomodulatory properties through the secretion of cytokines and exosomes and direct cell-cell interactions. MSC are routinely used in clinical trials and effectively resolve inflammatory conditions. Nevertheless, inconsistent clinical outcomes necessitate the need for more robust therapeutic phenotypes. The immunomodulatory properties of MSC can be enhanced and protracted by priming (aka licensing) them with IFN{gamma} and TNF. Yet these enhanced properties rapidly diminish, and prolonged stimulation could tolerize their response. Hence a balanced approach is needed to enhance the therapeutic potential of the MSC for consistent clinical performance. Here, we investigated the concentration-dependent effects of IFN{gamma} and TNF and developed gelatin-based microgels to sustain a licensed MSC phenotype. We show that IFN{gamma} treatment is more beneficial than TNF in promoting an immunomodulatory MSC phenotype. We also show that the microgels possess integrin-binding sites to support MSC attachment and a net positive charge to sequester the licensing cytokines electrostatically. Microgels are enzymatically degradable, and the rate is dependent on the enzyme concentration and matrix density. Our studies show that one milligram of microgels by dry mass can sequester up to 641 {+/-} 81 ng of IFN{gamma}. Upon enzymatic degradation, microgels exhibited a sustained release of IFN{gamma} that linearly correlated with their degradation rate. The MSC cultured on the IFN{gamma} sequestered microgels displayed efficient licensing potential comparable to or exceeding the effects of bolus IFN{gamma} treatment. When cultured with proinflammatory M1-like macrophages, the MSC-seeded on licensing microgel showed an enhanced immunomodulatory potential compared to untreated MSC and MSC treated with bolus IFN{gamma} treatment. Specifically, the MSC seeded on licensing microgels significantly upregulated Arg1, Mrc1, and Igf1, and downregulated Tnfa in M1-like macrophages compared to other treatment conditions. These licensing microgels are a potent immunomodulatory approach that shows substantial promise in elevating the efficacy of current MSC therapies and may find utility in treating chronic inflammatory conditions.

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.

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

1.Mesenchymal stem cell-derived exosome (MSC-EXO) transplantation has been suggested as an efficacious treatment to suppress spinal cord injury (SCI)-triggered neuroinflammation. However, an ethically acceptable method to continuously deliver MSC-EXOs to acute spinal lesions, without damaging nearby tissues/axons, has never been achieved. In this study, we fabricated a device comprising a patch containing MSCs and a microneedle array (MN-MSC patch) to treat severe SCI. When topically applied to an acute spinal lesion beneath the spinal dura, the soft microneedle (MN) array with reasonable mechanical strength avoided damaging the nearby spinal tissues, and the porous microstructure of MNs facilitated highly efficient MSC-EXO delivery. With the capacity for sustained delivery of MSC-EXOs, the MN-MSC patch was evaluated in a contusive rat SCI model. The MSCs encapsulated in the patch could survive for at least 7 days, encompassing the optimal time window for downregulating SCI-triggered neuroinflammation. As a result, MN-MSC patch treatment led to reduced cavity and scar tissue formation, greater angiogenesis, and improved survival of nearby tissues/axons. Remarkably, rats treated by this method achieved superior muscle control and exhibited robust hindlimb locomotion functional recovery. Conclusively, the MN-MSC patch device proposed here overcomes the current dilemma between treatment efficacy and ethical issues in treating acute SCI.

The reconstruction of critical-size bone defects in long bones remains a challenge for clinicians. We developed a new bioactive medical device for long bone repair by combining a 3D-printed architectured cylindrical scaffold made of clinical-grade polylactic acid (PLA) with a polyelectrolyte film coating delivering the osteogenic bone morphogenetic protein 2 (BMP-2). This film-coated scaffold was used to repair a sheep metatarsal 25-mm long critical-size bone defect. In vitro and in vivo biocompatibility of the film-coated PLA material were proved according to ISO standards. Scaffold geometry was found to influence BMP-2 incorporation. Bone regeneration was followed using X-ray scans, {micro}CT scans, and histology. We showed that scaffold internal geometry, notably pore shape, influenced bone regeneration, which was homogenous longitudinally. Scaffolds with cubic pores of [~]870 {micro}m and a low BMP-2 dose of [~]120 {micro}g/cm3 induced the best bone regeneration without any adverse effects. The visual score given by clinicians during animal follow-up was found to be an easy way to predict bone regeneration. This work opens perspectives for a clinical application in personalized bone regeneration.

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.

Articular cartilage repair is limited by the poor regenerative capacity of chondrocytes and their rapid dedifferentiation during in vitro expansion. This study investigated whether a decellularized and lyophilized cell-secreted matrix (CSM) could function as a bioactive material to regulate cell behavior, promote chondrogenic differentiation, and attenuate or reverse chondrocyte dedifferentiation without exogenous growth factor supplementation. CSM was generated from rabbit auricular perichondrial cells, decellularized, lyophilized, and characterized by histology, biochemical assays, and proteomic analysis. The resulting matrix was enriched in structurally and functionally relevant extracellular matrix proteins, including collagens, fibronectin, fibrillin, proteoglycans, and matricellular regulators, with minimal intracellular contamination and good batch-to-batch reproducibility. Functionally, CSM supported robust adhesion and proliferation of allogeneic and xenogeneic cells. Human articular chondrocytes cultured on CSM exhibited enhanced proliferation, sustained expression of cartilage-specific markers, and preserved type II collagen production over serial passages compared with standard plastic culture. CSM also promoted chondrogenic differentiation of human progenitor cells and partially reversed established chondrocyte dedifferentiation, as evidenced by increased expression of COL2A1, ACAN, SOX9, and COMP, with reduced COL1 expression and no induction of hypertrophic markers. These findings demonstrate that lyophilized CSM is a stable, off-the-shelf biomaterial capable of directing chondrocyte fate through intrinsic matrix-derived cues, highlighting its potential for cartilage tissue engineering and cell manufacturing applications.

Bone marrow mesenchymal stem cell (BM-MSC)-based implants is a promising method for bone regeneration. Implant failures are often caused by their susceptibility to bacterial infections. The aim of this study is to develop and evaluate antimicrobial scaffolds designed to protect BM-MSC-based implants from bacterial colonization while promoting bone repair. Biocompatible polycaprolactone (PCL) scaffolds were fabricated with incorporated antimicrobial agents, including silver nanoparticles (AgNPs) and vancomycin, using electrospinning and surface coating techniques. The scaffolds were characterized for morphology, mechanical properties, and antimicrobial release profiles. The findings of in vitro studies revealed that the scaffolds effectively inhibited bacterial growth (>90% reduction in CFUs) and biofilm formation for Staphylococcus aureus and Escherichia coli, without affecting BM-MSC viability or osteogenic potential. In vivo implantation on a rat femoral defect model showed that antimicrobial scaffolds significantly reduced bacterial load and enhanced bone regeneration, with micro-CT showing 65% bone volume compared to 35% in controls. Histological analysis confirmed active osteogenesis and infection control. These findings showed the potential of antimicrobial scaffolds as a dual-functional platform for bone tissue engineering. Future research can explore scaffold optimization for different applications and examine their efficacy against multi-drug-resistant bacteria to broaden clinical relevance.

Contemporary tissue engineering efforts often seek to use mesenchymal stem cells (MSCs) due to their potential to differentiate to various tissue-specific cells and generate a pro-regenerative secretome. While MSC differentiation and therapeutic potential can differ as a function of matrix environment, it may also be widely influenced as a function of donor-to-donor variability. Further, effects of passage number and donor sex may further convolute the identification of clinically effective MSC-mediated regeneration technologies. We report efforts to adapt a well-defined mineralized collagen scaffold platform to study the influence of MSC proliferation and osteogenic potential as a function of passage number and donor sex. Mineralized collagen scaffolds broadly support MSC osteogenic differentiation and regenerative potency in the absence of traditional osteogenic supplements for a wide range of MSCs (rabbit, rat, porcine, human). We obtained a library of bone marrow and adipose tissue derived stem cells to examine donor-variability of regenerative potency in mineralized collagen scaffolds. MSCs displayed reduced proliferative capacity as a function of passage duration. Further, MSCs showed significant sex-based differences. Notably, MSCs from male donors displayed significantly higher metabolic activity and proliferation while MSCs from female donor displayed significantly higher osteogenic response via increased alkaline phosphate activity, osteoprotegerin release, and mineral formation in vitro. Our study highlights the essentiality of considering MSC donor sex and culture expansion in future studies of biomaterial regenerative potential.