3D-Printed Antibacterial Scaffolds for the Regeneration of Alveolar Bone in Severe Periodontitis.

Current clinical treatment of periodontitis alleviates periodontal symptoms and helps to keep the disease under control for extended periods. Despite this, a significant destruction of the tooth's underlying bone tissue often takes place progressively. Herein, we present a two-way therapeutic approach for local delivery of antibacterial agents and bone tissue regeneration, incorporating ~1% w/w tetracycline hydrochloride (TCH) into a 3D-printed scaffold composed of poly(ε-caprolactone) (PCL). Samples were assessed for their morphological, physicochemical, pharmacokinetic, and antibacterial properties. Furthermore, osteoprecursor cells (MC3T3-E1) were employed to evaluate the osteoinductive potential of the drug-loaded scaffolds. Cell proliferation, viability, and differentiation were determined on all cell-seeded scaffolds. At the end of the culture, PCL-TCH scaffolds promoted abundant collagen organic matrix, demonstrating augmented alkaline phosphatase (ALP) activity and areas of accumulated mineralised bone tissue, despite their belayed cell proliferation. Based on the observed effectiveness of the PCL-TCH scaffolds to inhibit Staphylococcus aureus, these constructs could serve as an alternative bioactive implant that supports bacterial inhibition and favours a 3D microenvironment for bone tissue regeneration in severe periodontitis.

Protocol for Cell Colonization and Comprehensive Monitoring of Osteogenic Differentiation in 3D Scaffolds Using Biochemical Assays and Multiphoton Imaging.

The goal of bone tissue engineering is to build artificial bone tissue with properties that closely resemble human bone and thereby support the optimal integration of the constructs (biografts) into the body. The development of tissues in 3D scaffolds includes several complex steps that need to be optimized and monitored. In particular, cell-material interaction during seeding, cell proliferation and cell differentiation within the scaffold pores play a key role. In this work, we seeded two types of 3D-printed scaffolds with pre-osteoblastic MC3T3-E1 cells, proliferated and differentiated the cells, before testing and adapting different assays and imaging methods to monitor these processes. Alpha-TCP/HA (α-TCP with low calcium hydroxyapatite) and baghdadite (Ca3ZrSi2O9) scaffolds were used, which had comparable porosity (~50%) and pore sizes (~300-400 µm). Cell adhesion to both scaffolds showed ~95% seeding efficiency. Cell proliferation tests provided characteristic progression curves over time and increased values for α-TCP/HA. Transmitted light imaging displayed a homogeneous population of scaffold pores and allowed us to track their opening state for the supply of the inner scaffold regions by diffusion. Fluorescence labeling enabled us to image the arrangement and morphology of the cells within the pores. During three weeks of osteogenesis, ALP activity increased sharply in both scaffolds, but was again markedly increased in α-TCP/HA scaffolds. Multiphoton SHG and autofluorescence imaging were used to investigate the distribution, morphology, and arrangement of cells; collagen-I fiber networks; and hydroxyapatite crystals. The collagen-I networks became denser and more structured during osteogenic differentiation and appeared comparable in both scaffolds. However, imaging of the HA crystals showed a different morphology between the two scaffolds and appeared to arrange in the α-TCP/HA scaffolds along collagen-I fibers. ALP activity and SHG imaging indicated a pronounced osteo-inductive effect of baghdadite. This study describes a series of methods, in particular multiphoton imaging and complementary biochemical assays, to validly measure and track the development of bone tissue in 3D scaffolds. The results contribute to the understanding of cell colonization, growth, and differentiation, emphasizing the importance of optimal media supply of the inner scaffold regions.

Hypoxia Promotes Cartilage Regeneration in Cell-Seeded 3D-Printed Bioscaffolds Cultured with a Bespoke 3D Culture Device.

In this study, we investigated the effect of oxygen tension on the expansion of ADMSCs and on their differentiation toward their chondrocytic phenotype, regenerating a lab-based cartilaginous tissue with superior characteristics. Controversial results with reference to MSCs that were cultured under different hypoxic levels, mainly in 2D culturing settings combined with or without other biochemical stimulus factors, prompted our team to study the role of hypoxia on MSCs chondrogenic differentiation within an absolute 3D environment. Specifically, we used 3D-printed honeycomb-like PCL matrices seeded with ADMSCs in the presence or absence of TGF and cultured with a prototype 3D cell culture device, which was previously shown to favor nutrient/oxygen supply, cell adhesion, and infiltration within scaffolds. These conditions resulted in high-quality hyaline cartilage that was distributed uniformly within scaffolds. The presence of the TGF medium was necessary to successfully produce cartilaginous tissues with superior molecular and increased biomechanical properties. Despite hypoxia's beneficial effect, it was overall not enough to fully differentiate ADMSCs or even promote cell expansion within 3D scaffolds alone.

Specific immobilization of lipase on functionalized 3D printing scaffolds via enhanced hydrophobic interaction for efficient resolution of racemic 1-indanol.

Lipase immobilization with hydrophobic interaction is of interesting exploration, and some functionalized groups on supports are special for activity increasing. To achieved a good performance of cost-effective immobilization on macro-supports for feasible usage and recycle, eco-friendly PLA-based 3D printing macro-scaffolds with fabrication was designed, and phenyl groups with different length of linkers and combined two kinds of groups were anchored for lipase YCJ01 binding with improving payload, the highest enzyme expression of 2227.5 U/g, activity recovery of 137.3%, and increasing specific activity of 815.9 U/mg were attained by using PLA@AMTS-C7-Ph/PLA@AMTS-C9-Ph scaffolds as carries. The immobilized lipase YCJ01 on bifunctionalized 3D printing scaffolds was further applied to the efficient resolution of racemic 1-indanol (267 mM) with high stereoselectivity using a binary solvent system. The immobilized lipase YCJ01 could control the over transesterification of (S)-1-indanol and exhibit good operational stability of repetitive usage for 9 cycles. This is beneficial to obtain the high enantiomerical pure product by feasible separation of immobilized biocatalyst without rigorous operation.

Engineering 2D Mesoporous Silica@MXene-Integrated 3D-Printing Scaffolds for Combinatory Osteosarcoma Therapy and NO-Augmented Bone Regeneration.

The rising concerns of the recurrence and bone deficiency in surgical treatment of malignant bone tumors have raised an urgent need of the advance of multifunctional therapeutic platforms for efficient tumor therapy and bone regeneration. Herein, the construction of a multifunctional biomaterial system is reported by the integration of 2D Nb2 C MXene wrapped with S-nitrosothiol (RSNO)-grafted mesoporous silica with 3D-printing bioactive glass (BG) scaffolds (MBS). The near infrared (NIR)-triggered photonic hyperthermia of MXene in the NIR-II biowindow and precisely controlled nitric oxide (NO) release are coordinated for multitarget ablation of bone tumors to enhance localized osteosarcoma treatment. The in situ formed phosphorus and calcium components degraded from BG scaffold promote bone-regeneration bioactivity, augmented by sufficient blood supply triggered by on-demand NO release. The tunable NO generation plays a crucial role in sequential adjuvant tumor ablation, combinatory promotion of coupled vascularization, and bone regeneration. This study demonstrates a combinatory osteosarcoma ablation and a full osseous regeneration as enabled by the implantation of MBS. The design of multifunctional scaffolds with the specific features of controllable NO release, highly efficient photothermal conversion, and stimulatory bone regeneration provides an intriguing biomaterial platform for the diversified treatment of bone tumors.

Osteoblast and osteoclast activity on collagen-based 3D printed scaffolds enriched with strontium-doped bioactive glasses and hydroxyapatite nanorods for bone tissue engineering.

Bone tissue engineering (BTE) aims to promote bone regeneration by means of the synergistic effect of biomaterials, cells, and other factors, as potential alternative to conventional treatments for bone fractures. To this aim, a composite material was developed, based on collagen type I, strontium-enriched mesoporous bioactive glasses, and hydroxyapatite nanorods as bioactive and biomimetic components. Nanostructured scaffolds were 3D printed and subsequently chemically crosslinked with genipin to improve mechanical properties and stability. The developed nanostructured system was maintained in culture until 3 weeks with a co-culture of human bone cells to provide anex vivomodel of bone microenvironment and examine the cellular crosstalk and signaling pathways through paracrine cell activities. Human osteoblasts (OBs), derived from trabecular bone, and human osteoclast precursors (OCs), isolated from buffy coat samples were involved, with OBs seeded on the scaffold and OC precursors seeded in a transwell device. When compared to the material without inorganic components, the bioactive and biomimetic scaffold positively influenced cell proliferation and cell metabolic activity, boosting alkaline phosphatase activity of OBs, and reducing OC differentiation. Thus, the bioactive and biomimetic system promoted an enhanced cellular response, highlighting its potential application in BTE.

Defining the immunological compatibility of graphene oxide-loaded PLGA scaffolds for biomedical applications.

Graphene oxide (GO), a carbon-based nanomaterial, presents significant potential across biomedical fields such as bioimaging, drug delivery, biosensors, and phototherapy. This study examines the effects of integrating GO into poly(lactic-co-glycolic acid) (PLGA) scaffolds on human immune cell function. Our results demonstrate that high concentrations of GO reduce the viability of peripheral blood mononuclear cells (PBMCs) following stimulation with anti-CD3 antibody. This reduction extends to T lymphocyte activation, evident from the diminished proliferative response to T cell receptor engagement and impaired differentiation into T helper subsets and regulatory T cells. Interestingly, although GO induces a minimal response in resting monocytes, but it significantly affects both the viability and the differentiation potential of monocytes induced to mature toward M1 pro-inflammatory and M2-like immunoregulatory macrophages. This study seeks to address a critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating various concentrations of GO on primary immune cells, specifically PBMCs isolated from healthy donors. Our findings emphasize the need to optimize the GO to PLGA ratios and scaffold design to advance PLGA-GO-based biomedical applications. STATEMENT OF SIGNIFICANCE: Graphene oxide (GO) holds immense promise for biomedical applications due to its unique properties. However, concerns regarding its potential to trigger adverse immune responses remain. This study addresses this critical gap by investigating the in vitro immunomodulatory effects of PLGA scaffolds incorporating increasing GO concentrations on human peripheral blood mononuclear cells (PBMCs). By elucidating the impact on cell viability, T cell proliferation and differentiation, and the maturation/polarization of antigen-presenting cells, this work offers valuable insights for designing safe and immunologically compatible GO-based biomaterials for future clinical translation.

Fabrication and properties of PLA/ß-TCP scaffolds using liquid crystal display (LCD) photocuring 3D printing for bone tissue engineering.

Introduction: Bone defects remain a thorny challenge that clinicians have to face. At present, scaffolds prepared by 3D printing are increasingly used in the field of bone tissue repair. Polylactic acid (PLA) has good thermoplasticity, processability, biocompatibility, and biodegradability, but the PLA is brittle and has poor osteogenic performance. Beta-tricalcium phosphate (ß-TCP) has good mechanical properties and osteogenic induction properties, which can make up for the drawbacks of PLA. Methods: In this study, photocurable biodegradable polylactic acid (bio-PLA) was utilized as the raw material to prepare PLA/ß-TCP slurries with varying ß-TCP contents (ß-TCP dosage at 0%, 10%, 20%, 30%, 35% of the PLA dosage, respectively). The PLA/ß-TCP scaffolds were fabricated using liquid crystal display (LCD) light-curing 3D printing technology. The characterization of the scaffolds was assessed, and the biological activity of the scaffold with the optimal compressive strength was evaluated. The biocompatibility of the scaffold was assessed through CCK-8 assays, hemocompatibility assay and live-dead staining experiments. The osteogenic differentiation capacity of the scaffold on MC3T3-E1 cells was evaluated through alizarin red staining, alkaline phosphatase (ALP) detection, immunofluorescence experiments, and RT-qPCR assays. Results: The prepared scaffold possesses a three-dimensional network structure, and with an increase in the quantity of ß-TCP, more ß-TCP particles adhere to the scaffold surface. The compressive strength of PLA/ß-TCP scaffolds exhibits a trend of initial increase followed by decrease with an increasing amount of ß-TCP, reaching a maximum value of 52.1 MPa at a 10% ß-TCP content. Degradation rate curve results indicate that with the passage of time, the degradation rate of the scaffold gradually increases, and the pH of the scaffold during degradation shows an alkaline tendency. Additionally, Live/dead staining and blood compatibility experiments suggest that the prepared PLA/ß-TCP scaffold demonstrates excellent biocompatibility. CCK-8 experiments indicate that the PLA/ß-TCP group promotes cell proliferation, and the prepared PLA/ß-TCP scaffold exhibits a significant ability to enhance the osteogenic differentiation of MC3T3-E1 cells in vitro. Discussion: 3D printed LCD photocuring PLA/ß-TCP scaffolds could improve surface bioactivity and lead to better osteogenesis, which may provide a unique strategy for developing bioactive implants in orthopedic applications.

Slow and steady wins the race: Fractionated near-infrared treatment empowered by graphene-enhanced 3D scaffolds for precision oncology.

Surgically addressing tumors poses a challenge, requiring a tailored, multidisciplinary approach for each patient based on the unique aspects of their case. Innovative therapeutic regimens combined to reliable reconstructive methods can contribute to an extended patient's life expectancy. This study presents a detailed comparative investigation of near-infrared therapy protocols, examining the impact of non-fractionated and fractionated irradiation regimens on cancer treatment. The therapy is based on the implantation of graphene oxide/poly(lactic-co-glycolic acid) three-dimensional printed scaffolds, exploring their versatile applications in oncology by the examination of pro-inflammatory cytokine secretion, immune response, and in vitro and in vivo tumor therapy. The investigation into cell death patterns (apoptosis vs necrosis) underlines the pivotal role of protocol selection underscores the critical influence of treatment duration on cell fate, establishing a crucial parameter in therapeutic decision-making. In vivo experiments corroborated the profound impact of protocol selection on tumor response. The fractionated regimen emerged as the standout performer, achieving a substantial reduction in tumor size over time, surpassing the efficacy of the non-fractionated approach. Additionally, the fractionated regimen exhibited efficacy also in targeting tumors in proximity but not in direct contact to the scaffolds. Our results address a critical gap in current research, highlighting the absence of a standardized protocol for optimizing the outcome of photodynamic therapy. The findings underscore the importance of personalized treatment strategies in achieving optimal therapeutic efficacy for precision cancer therapy.

Mechanical and in vitro study of 3D printed silk fibroin and bone-based composites biomaterials for bone implant application.

When treating orthopaedic damage or illness and accidental fracture, bone grafting remains the gold standard of treatment. In cases where this approach does not seem achievable, bone tissue engineering can offer scaffolding as a substitute. Defective and fractured bone tissue is extracted and substituted with porous scaffold structures to aid in the process of tissue regeneration. 3D bioprinting has demonstrated enormous promise in recent years for producing scaffold structures with the necessary capabilities. In order to create composite biomaterial inks for 3D bioprinting, three different materials were combined such as silk fibroin, bone particles, and synthetic biopolymer poly (ε-caprolactone) (PCL). These biomaterials were used to fabricate the two composites scaffolds such as: silk fibroin + bovine bone (SFB) and silk fibroin + bovine bone + Polycaprolactone (SFBP). The biomechanical, structural, and biological elements of the manufactured composite scaffolds were characterized in order to determine their suitability as a possible biomaterial for the production of bone tissue. The in vitro bioactivity of the two composite scaffolds was assessed in the simulated body fluids, and the swelling and degradation characteristics of the two developed scaffolds were analyzed separately over time. The results showed that the mechanical durability of the composite scaffolds was enhanced by the bovine bone particles, up to a specific concentration in the silk fibroin matrix. Furthermore, the incorporation of bone particles improved the bioactive composite scaffolds' capacity to generate hydroxyapatite in vitro. The combined findings show that the two 3D printed bio-composites scaffolds have the required mechanical strength and may be applied to regeneration of bone tissue and restoration, since they resemble the characteristics of native bone.

Synthetic Bone Blocks Produced by Additive Manufacturing in the Repair of Critical Bone Defects.

This study evaluated the efficacy of synthetic bone blocks, composed of hydroxyapatite (HA) or ß-tricalcium phosphate (B-TCP), which were produced by additive manufacturing and used for the repair of critical size bone defects (CSDs) in rat calvaria. Sixty rats were divided into five groups (n = 12): blood clot (CONTROL), 3D-printed HA (HA), 3D-printed ß-TCP (B-TCP), 3D-printed HA + autologous micrograft (HA+RIG), and 3D-printed ß-TCP + autologous micrograft (B-TCP+RIG). CSDs were surgically created in the parietal bone and treated with the respective biomaterials. The animals were euthanized at 30 and 60 days postsurgery for microcomputed tomography (micro-CT) histomorphometric, and immunohistochemical analysis to assess new bone formation. Micro-CT analysis showed that both biomaterials were incorporated into the animals' calvaria. The HA+RIG group, especially at 60 days, exhibited a significant increase in bone formation compared with the control. The use of 3D-printed bioceramics resulted in thinner trabeculae but a higher number of trabeculae compared with the control. Histomorphometric analysis showed bone islands in close contact with the B-TCP and HA blocks at 30 days. The HA blocks (HA and HA+RIG groups) showed statistically higher new bone formation values with further improvement when autologous micrografts were included. Immunohistochemical analysis showed the expression of bone repair proteins. At 30 days, the HA+RIG group had moderate Osteopontin (OPN) staining, indicating that the repair process had started, whereas other groups showed no staining. At 60 days, the HA+RIG group showed slight staining, similar to that of the control. Osteocalcin (OCN) staining, indicating osteoblastic activity, showed moderate expression in the HA and HA+RIG groups at 30 days, with slight expression in the B-TCP and B-TCP+RIG groups. The combination of HA blocks with autologous micrografts significantly enhanced bone repair, suggesting that the presence of progenitor cells and growth factors in the micrografts contributed to the improved outcomes. It was concluded that 3D-printed bone substitute blocks, associated with autologous micrografts, are highly effective in promoting bone repair in CSDs in rat calvaria.

Boron substitution in silicate bioactive glass scaffolds to enhance bone differentiation and regeneration.

Commercially available bioactive glasses (BAGs) are exclusively used in powder form, due to their tendency to crystallize. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. This study investigates the potential of 1393B20 scaffolds to support bone regeneration and mineralization in vitro and in vivo, in comparison to silicate 1393. Both scaffolds supported human adipose stem cells proliferation, either in direct contact for the 1393, or mainly around for the 1393B20. Similarly, both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. In addition, these scaffolds supported bone regeneration and osteoclast/osteoblast activity in vivo in critical-sized rat calvarial defect. Nevertheless, mineralization and collagen formation were significantly enhanced for the 1393B20, at 3-months post-implantation, assigned to faster and more complete dissolution of the scaffolds. Thus, 1393B20 demonstrates greater promise for bone tissue engineering certainly due to its time-controlled release of boron and silicon. STATEMENT OF SIGNIFICANCE: Bioactive glasses (BAGs) show great promise in bone tissue engineering as they effectively bond with bone tissue, fostering integration and regeneration. Silicate BAG 1393 was developed to allow fiber drawing and scaffold sintering, but its slow degradation limits its potential. To enable scaffold manufacturing while maintaining glass dissolution rate close to that of commercially available BAGs, the borosilicate glass 1393B20 was developed. Both BAGs induced osteogenesis and angiogenesis in vitro, with a better pro-angiogenic influence of the 1393B20. The presence of boron in the 1393B20 enhanced mineralization and collagen formation in vivo compared to 1393, probably due to its faster dissolution rate. Here, 1393B20 demonstrated greater promise for bone tissue engineering compared to the well-known 1393 BAG.

Ti3C2Tx MXene-Decorated 3D-Printed Ceramic Scaffolds for Enhancing Osteogenesis by Spatiotemporally Orchestrating Inflammatory and Bone Repair Responses.

Inflammatory responses play a central role in coordinating biomaterial-mediated tissue regeneration. However, precise modulation of dynamic variations in microenvironmental inflammation post-implantation remains challenging. In this study, the traditional ß-tricalcium phosphate-based scaffold is remodeled via ultrathin MXene-Ti3C2 decoration and Zn2+/Sr2+ ion-substitution, endowing the scaffold with excellent reactive oxygen species-scavenging ability, near-infrared responsivity, and enhanced mechanical properties. The induction of mild hyperthermia around the implant via periodic near-infrared irradiation facilitates spatiotemporal regulation of inflammatory cytokines secreted by a spectrum of macrophage phenotypes. The process initially amplifies the pro-inflammatory response, then accelerates M1-to-M2 macrophage polarization transition, yielding a satisfactory pattern of osteo-immunomodulation during the natural bone healing process. Later, sustained release of Zn2+/Sr2+ ions with gradual degradation of the 3D scaffold maintains the favorable reparative M2-dominated immunological microenvironment that supports new bone mineralization. Precise temporal immunoregulation of the bone healing process by the intelligent 3D scaffold enhances bone regeneration in a rat cranial defect model. This strategy paves the way for the application of ß-tricalcium phosphate-based materials to guide the dynamic inflammatory and bone tissue responses toward a favorable outcome, making clinical treatment more predictable and durable. The findings also demonstrate that near-infrared irradiation-derived mild hyperthermia is a promising method of immunomodulation.

3D-printed near-infrared-light-responsive on-demand drug-delivery scaffold for bone regeneration.

Although several bioactive 3D-printed bone scaffolds loaded with multiple kinds of biomolecules for enhanced bone regeneration have been recently developed, the manipulation of on-demand release profiles of different biomolecules during bone regeneration remains challenging. Herein, a 3D-printed dual-drug-loaded biomimetic scaffold to regulate the host stem cell recruitment and osteogenic differentiation in a two-stage process for bone regeneration was successfully fabricated. First, a chemotactic small-molecule drug, namely, simvastatin (SIM) was directly incorporated into the hydroxyapatite/collagen bioink for printing and could be rapidly released during the early stage of bone regeneration. Further, near-infrared (NIR)-light-responsive polydopamine-coated hydroxyapatite nanoparticles were designed to deliver the osteogenic drug, i.e., pargyline (PGL) in a controllable manner. Together, our scaffold displayed an on-demand sequential release of those two drugs and could optimize their therapeutic effects to align with the stem cell recruitment and osteoblastic differentiation, thereby promoting bone regeneration. The results confirmed the suitable mechanical strength, high photothermal conversion efficiency, good biocompatibility of our scaffold. The scaffold loaded with SIM could efficiently accelerate the migration of stem cells. In addition, the scaffold with on-demand sequential release promoted alkaline phosphatase (ALP) activity, significantly upregulated gene expression levels of osteogenesis-related markers, and enhanced new-bone-formation capabilities in rabbit cranial defect models. Altogether, this scaffold not only offers a promising strategy to control the behavior of stem cells during bone regeneration but also provides an efficient strategy for controllable sequential release of different biomolecule in bone tissue engineering.

Platelet-rich plasma in orthopedics: Bridging innovation and clinical applications for bone repair.

In the burgeoning domain of orthopedic therapeutic research, Platelet-Rich Plasma (PRP) has firmly established its position, transforming paradigms ranging from tissue regeneration to the management of chondral lesions. This review delves into PRP's recent integrations with cutting-edge interventions such as 3D-printed scaffolds, its role in bone and cartilage defect management, and its enhanced efficacy when combined with molecules like Kartogenin (KGN) for fibrocartilage zone repair. Significant attention is paid to tissue engineering for meniscal interventions, where a combination of KGN, PRP, and bone marrow-derived mesenchymal stem cells are under exploration. Within the sphere of osteochondral regenerative therapy, the synergy of PRP with Bone Marrow Aspirate Concentrate (BMAC) represents a noteworthy leap towards cartilage regeneration. The innovative incorporation of PRP with biomaterials like hydroxyapatite and graphene oxide further underscores its versatility in supporting structural integrity and ensuring sustained growth factor release. However, while PRP's autologous and nontoxic nature makes it an inherently safe option, concerns arising from its preparation methods, particularly with bovine thrombin, necessitate caution. As of 2023, despite the burgeoning promise of PRP in bone healing, the quest for its standardization, optimization, and substantiation through rigorous clinical trials continues. This comprehensive review elucidates the contemporary applications, challenges, and future trajectories of PRP in orthopedics, aiming to spotlight areas primed for further research and exploration.

Cell Instructive Behavior of Composite Scaffolds in a Co-Culture of Human Mesenchymal Stem Cells and Peripheral Blood Mononuclear Cells.

The in vitro evaluation of 3D scaffolds for bone tissue engineering in mono-cultures is a common practice; however, it does not represent the native complex nature of bone tissue. Co-cultures of osteoblasts and osteoclasts, without the addition of stimulating agents for monitoring cellular cross-talk, remains a challenge. In this study, a growth factor-free co-culture of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) and human peripheral blood mononuclear cells (hPBMCs) has been established and used for the evaluation of 3D-printed scaffolds for bone tissue engineering. The scaffolds were produced from PLLA/PCL/PHBV polymeric blends, with two composite materials produced through the addition of 2.5% w/v nanohydroxyapatite (nHA) or strontium-substituted nanohydroxyapatite (Sr-nHA). Cell morphology data showed that hPBMCs remained undifferentiated in co-culture, while no obvious differences were observed in the mono- and co-cultures of hBM-MSCs. A significantly increased alkaline phosphatase (ALP) activity and osteogenic gene expression was observed in co-culture on Sr-nHA-containing scaffolds. Tartrate-resistant acid phosphatase (TRAP) activity and osteoclastogenic gene expression displayed significantly suppressed levels in co-culture on Sr-nHA-containing scaffolds. Interestingly, mono-cultures of hPBMCs on Sr-nHA-containing scaffolds indicated a delay in osteoclasts formation, as evidenced from TRAP activity and gene expression, demonstrating that strontium acts as an osteoclastogenesis inhibitor. This co-culture study presents an effective 3D model to evaluate the regenerative capacity of scaffolds for bone tissue engineering, thus minimizing time-consuming and costly in vivo experiments.

Release Kinetics of Metronidazole from 3D Printed Silicone Scaffolds for Sustained Application to the Female Reproductive Tract.

Sustained vaginal administration of antibiotics or probiotics has been proposed to improve treatment efficacy for bacterial vaginosis. 3D printing has shown promise for development of systems for local agent delivery. In contrast to oral ingestion, agent release kinetics can be fine-tuned by the 3D printing of specialized scaffold designs tailored for particular treatments while enhancing dosage effectiveness via localized sustained release. It has been challenging to establish scaffold properties as a function of fabrication parameters to obtain sustained release. In particular, the relationships between scaffold curing conditions, compressive strength, and drug release kinetics remain poorly understood. This study evaluates 3D printed scaffold formulation and feasibility to sustain the release of metronidazole, a commonly used antibiotic for BV. Cylindrical silicone scaffolds were printed and cured using three different conditions relevant to potential future incorporation of temperature-sensitive labile biologics. Compressive strength and drug release were monitored for 14d in simulated vaginal fluid to assess long-term effects of fabrication conditions on mechanical integrity and release kinetics. Scaffolds were mechanically evaluated to determine compressive and tensile strength, and elastic modulus. Release profiles were fitted to previous kinetic models to differentiate potential release mechanisms. The Higuchi, Korsmeyer-Peppas, and Peppas-Sahlin models best described the release, indicating similarity to release from insoluble or polymeric matrices. This study shows the feasibility of 3D printed silicone scaffolds to provide sustained metronidazole release over 14d, with compressive strength and drug release kinetics tuned by the fabrication parameters.

Development of an All-Marine 3D Printed Bioactive Hydrogel Dressing for Treatment of Hard-to-Heal Wounds.

Current standard wound care involves dressings that provide moisture and protection; however, dressings providing active healing are still scarce and expensive. We aimed to develop an ecologically sustainable 3D printed bioactive hydrogel-based topical wound dressing targeting healing of hard-to-heal wounds, such as chronic or burn wounds, which are low on exudate. To this end, we developed a formulation composed of renewable marine components; purified extract from unfertilized salmon roe (heat-treated X, HTX), alginate from brown seaweed, and nanocellulose from tunicates. HTX is believed to facilitate the wound healing process. The components were successfully formulated into a 3D printable ink that was used to create a hydrogel lattice structure. The 3D printed hydrogel showed a HTX release profile enhancing pro-collagen I alpha 1 production in cell culture with potential of promoting wound closure rates. The dressing has recently been tested on burn wounds in Göttingen minipigs and shows accelerated wound closure and reduced inflammation. This paper describes the dressings development, mechanical properties, bioactivity, and safety.

An injectable and 3D printable pro-chondrogenic hyaluronic acid and collagen type II composite hydrogel for the repair of articular cartilage defects.

Current treatments for repairing articular cartilage defects are limited. However, pro-chondrogenic hydrogels formulated using articular cartilage matrix components (such as hyaluronic acid (HA) and collagen type II (Col II)), offer a potential solution if they could be injected into the defect via minimally invasive arthroscopic procedures, or used as bioinks to 3D print patient-specific customised regenerative scaffolds-potentially combined with cells. However, HA and Col II are difficult to incorporate into injectable/3D printable hydrogels due to poor physicochemical properties. This study aimed to overcome this by developing an articular cartilage matrix-inspired pro-chondrogenic hydrogel with improved physicochemical properties for both injectable and 3D printing (3DP) applications. To achieve this, HA was methacrylated to improve mechanical properties and mixed in a 1:1 ratio with Col I, a Col I/Col II blend or Col II. Col I possesses superior mechanical properties to Col II and so was hypothesised to enhance hydrogel mechanical properties. Rheological analysis showed that the pre-gels had viscoelastic and shear thinning properties. Subsequent physicochemical analysis of the crosslinked hydrogels showed that Col II inclusion resulted in a more swollen and softer polymer network, without affecting degradation time. While all hydrogels exhibited exemplary injectability, only the Col I-containing hydrogels had sufficient mechanical stability for 3DP applications. To facilitate 3DP of multi-layered scaffolds using methacrylated HA (MeHA)-Col I and MeHA-Col I/Col II, additional mechanical support in the form of a gelatin slurry support bath freeform reversible embedding of suspended hydrogels was utilised. Biological analysis revealed that Col II inclusion enhanced hydrogel-embedded MSC chondrogenesis, thus MeHA-Col II was selected as the optimal injectable hydrogel, and MeHA-Col I/Col II as the preferred bioink. In summary, this study demonstrates how tailoring biomaterial composition and physicochemical properties enables development of pro-chondrogenic hydrogels with potential for minimally invasive delivery to injured articular joints or 3DP of customised regenerative implants for cartilage repair.

Regulation of Osteoimmune Microenvironment and Osteogenesis by 3D-Printed PLAG/black Phosphorus Scaffolds for Bone Regeneration.

The treatment of bone defects remains a significant challenge to be solved clinically. Immunomodulatory properties of orthopedic biomaterials have significance in regulating osteoimmune microenvironment for osteogenesis. A lactic acid-co-glycolic acid (PLGA) scaffold incorporates black phosphorus (BP) fabricated by 3D printing technology to investigate the effect of BP on osteoimmunomodulation and osteogenesis in site. The PLGA/BP scaffold exhibits suitable biocompatibility, biodegradability, and mechanical properties as an excellent microenvironment to support new bone formation. The studies' result also demonstrate that the PLGA/BP scaffolds are able to recruit and stimulate macrophages M2 polarization, inhibit inflammation, and promote human bone marrow mesenchymal stem cells (hBMSCs) proliferation and differentiation, which in turn promotes bone regeneration in the distal femoral defect region of steroid-associated osteonecrosis (SAON) rat model. Moreover, it is screened and demonstrated that PLGA/BP scaffolds can promote osteogenic differentiation by transcriptomic analysis, and PLGA/BP scaffolds promote osteogenic differentiation and mineralization by activating PI3K-AKT signaling pathway in hBMSC cells. In this study, it is shown that the innovative PLGA/BP scaffolds are extremely effective in stimulating bone regeneration by regulating macrophage M2 polarization and a new strategy for the development of biomaterials that can be used to repair bone defects is offered.