Development and application of hydroxyapatite-based scaffolds for bone tissue regeneration: A systematic literature review.

Hydroxyapatite [HA, Ca10(PO4)6(OH)2], with its robust biocompatibility and bioactivity, has found extensive utility in bone grafting, replacement therapies, and supplemental medical materials. HA is highly regarded for its osteoconductive properties because it boasts hydrophilicity, nontoxicity, non-allergenicity, and non-mutagenicity. Nevertheless, HA's intrinsic mechanical weakness has spurred efforts to enhance its properties. This enhancement is achieved through ion incorporation, with elements such as magnesium, zinc, lithium, strontium, boron, and others being integrated into the HA structure. In the domain of orthopedics, HA-based scaffolds have emerged as a solution for addressing prevalent issues like bone deformities and defects stemming from congenital anomalies, injuries, trauma, infections, or tumors. The fabrication of three-dimensional scaffolds (3D scaffolds) has enabled advancements in bone regeneration and replacement, with a focus on practical applications such as repairing calvarial, skull, and femoral defects. In vitro and in vivo assessments have substantiated the effectiveness of 3D scaffolds for bone defect repair, regeneration, and tissue engineering. Beyond bone-related applications, scaffolds demonstrate versatility in enhancing cartilage healing and serving as bioimplants. The wide array of scaffold applications underscores their ongoing potential for further development in the realm of medical science.

Bionic Bilayer Scaffold for Synchronous Hyperthermia Therapy of Orthotopic Osteosarcoma and Osteochondral Regeneration.

Large osseous void, postsurgical neoplastic recurrence, and slow bone-cartilage repair rate raise an imperative need to develop functional scaffold in clinical osteosarcoma treatment. Herein, a bionic bilayer scaffold constituting croconaine dye-polyethylene glycol@sodium alginate hydrogel and poly(l-lactide)/hydroxyapatite polymer matrix is fabricated to simultaneously achieve a highly efficient killing of osteosarcoma and an accelerated osteochondral regeneration. First, biomimetic osteochondral structure along with adequate interfacial interaction of the bilayer scaffold provide a structural reinforcement for transverse osseointegration and osteochondral regeneration, as evidenced by upregulated specific expressions of collagen type-I, osteopontin, and runt-related transcription factor 2. Meanwhile, thermal ablation of the synthesized nanoparticles and mitochondrial dysfunction caused by continuously released hydroxyapatite induce residual tumor necrosis synergistically. To validate the capabilities of inhibiting tumor growth and promoting osteochondral regeneration of our proposed scaffold, a novel orthotopic osteosarcoma model simulating clinical treatment scenarios of bone tumors is established on rats. Based on amounts of in vitro and in vivo results, an effective killing of osteosarcoma and a suitable osteal-microenvironment modulation of such bionic bilayer composite scaffold are achieved, which provides insightful implications for photonic hyperthermia therapy against osteosarcoma and following osseous tissue regeneration.

Tissue engineered periosteum: Fabrication of a gelatin basedtrilayer composite scaffold with biomimetic properties for enhanced bone healing.

The periosteum, a vascularized tissue membrane, is essential in bone regeneration following fractures and bone loss due to some other reasons, yet there exist several research gaps concerning its regeneration. These gaps encompass reduced cellular proliferation and bioactivity, potential toxicity, heightened stiffness of scaffold materials, unfavorable porosity, expensive materials and procedures, and suboptimal survivability or inappropriate degradation rates of the implanted materials. This research used an interdisciplinary approach by forming a new material fabricated through electrospinning for the proposed application as a layer-by-layer tissue-engineered periosteum (TEP). TEP comprises poly(ε-caprolactone) (PCL), PCL/gelatin/magnesium-doped zinc oxide (vascular layer), and gelatin/bioactive glass/COD liver oil (osteoconductive layer). These materials were selected for their diverse properties, when integrated into the scaffold formation, successfully mimic the characteristics of native periosteum. Scanning electron microscopy (SEM) was employed to confirm the trilayer structure of the scaffold and determine the average fiber diameter. In-vitro degradation and swelling studies demonstrated a uniform degradation rate that matches the typical recovery time of periosteum. The scaffold exhibited excellent mechanical properties comparable to natural periosteum. Furthermore, the sustained release kinetics of COD liver oil were observed in the trilayer scaffold. Cell culture results indicated that the three-dimensional topography of the scaffold promoted cell growth, proliferation, and attachment, confirming its non-toxicity, biocompatibility, and bioactivity. This study suggests that the fabricated scaffold holds promise as a potential artificial periosteum for treating periostitis and bone fractures.

Chondrogenesis of mesenchymal stromal cells on the 3D printed polycaprolactone/fibrin/decellular cartilage matrix hybrid scaffolds in the presence of piascledine.

Nowadays, cartilage tissue engineering (CTE) is considered important due to lack of repair of cartilaginous lesions and the absence of appropriate methods for treatment. In this study, polycaprolactone (PCL) scaffolds were fabricated by three-dimensional (3D) printing and were then coated with fibrin (F) and acellular solubilized extracellular matrix (ECM). After extracting adipose-derived stem cells (ADSCs), 3D-printed scaffolds were characterized and compared to hydrogel groups. After inducing the chondrogenic differentiation in the presence of Piascledine and comparing it with TGF-ß3 for 28 days, the expression of genes involved in chondrogenesis (AGG, COLII) and the expression of the hypertrophic gene (COLX) were examined by real-time PCR. The expression of proteins COLII and COLX was also determined by immunohistochemistry. Glycosaminoglycan was measured by toluidine blue staining. 3D-printed scaffolds clearly improved cell proliferation, viability, water absorption and compressive strength compared to the hydrogel groups. Moreover, the use of compounds such as ECM and Piascledine in the process of ADSCs chondrogenesis induction increased cartilage-specific markers and decreased the hypertrophic marker compared to TGF-ß3. In Piascledine groups, the expression of COLL II protein, COLL II and Aggrecan genes, and the amount of glycosaminoglycan showed a significant increase in the PCL/F/ECM compared to the PCL and PCL/F groups.

Reduced graphene oxide-reinforced tricalcium phosphate/gelatin/chitosan light-responsive scaffolds for application in bone regeneration.

Bone is a mineralized tissue with the intrinsic capacity for constant remodeling. Rapid prototyping techniques, using biomaterials that mimic the bone native matrix, have been used to develop osteoinductive and osteogenic personalized 3D structures, which can be further combined with drug delivery and phototherapy. Herein, a Fab@Home 3D Plotter printer was used to promote the layer-by-layer deposition of a composite mixture of gelatin, chitosan, tricalcium phosphate, and reduced graphene oxide (rGO). The phototherapeutic potential of the new NIR-responsive 3D_rGO scaffolds was assessed by comparing scaffolds with different rGO concentrations (1, 2, and 4 mg/mL). The data obtained show that the rGO incorporation confers to the scaffolds the capacity to interact with NIR light and induce a hyperthermy effect, with a maximum temperature increase of 16.7 °C after under NIR irradiation (10 min). Also, the increase in the rGO content improved the hydrophilicity and mechanical resistance of the scaffolds, particularly in the 3D_rGO4. Furthermore, the rGO could confer an NIR-triggered antibacterial effect to the 3D scaffolds, without compromising the osteoblasts' proliferation and viability. In general, the obtained data support the development of 3D_rGO for being applied as temporary scaffolds supporting the new bone tissue formation and avoiding the establishment of bacterial infections.

3D bioprinting of Salvianolic acid B-sodium alginate-gelatin skin scaffolds promotes diabetic wound repair via antioxidant, anti-inflammatory, and proangiogenic effects.

In patients with diabetic wounds, wound healing is impaired due to the presence of persistent oxidative stress, an altered inflammatory response, and impaired angiogenesis and epithelization. Salvianolic acid B (SAB), which is derived from the Chinese medicinal plant Salvia miltiorrhiza, has been found to exhibit antioxidant, anti-inflammatory, and proangiogenic effects. Previous studies have used 3D bioprinting technology incorporating sodium alginate (SA) and gelatin (Gel) as basic biomaterials to successfully produce artificial skin. In the current study, 3D bioprinting technology was used to incorporate SAB into SA-Gel to form a novel SAB-SA-Gel composite porous scaffold. The morphological characteristics, physicochemical characteristics, biocompatibility, and SAB release profile of the SAB-SA-Gel scaffolds were evaluated in vitro. In addition, the antioxidant, anti-inflammatory, and proangiogenic abilities of the SAB-SA-Gel scaffolds were evaluated in cells and in a rat model. Analysis demonstrated that 1.0 wt% (the percentage of SAB in the total weight of the solution containing SA and Gel) SAB-SA-Gel scaffolds had strong antioxidant, anti-inflammatory, and proangiogenic properties both in cells and in the rat model. The 1.0% SAB-SA-Gel scaffold reduced the expression of tumor necrosis factor-α, interleukin-6, and interluekin-1ß and increased the expression of transforming growth factor-ß. In addition, this scaffold removed excessive reactive oxygen species by increasing the expression of superoxide dismutase, thereby protecting fibroblasts from injury. The scaffold increased the expression of vascular endothelial growth factor and platelet/endothelial cell adhesion molecule-1, accelerated granulation tissue regeneration and collagen deposition, and promoted wound healing. These findings suggest that this innovative scaffold may have promise as a simple and efficient approach to managing diabetic wound repair.

In vitro assessment of Momordica charantia/Hypericum perforatum oils loaded PCL/Collagen fibers: Novel scaffold for tissue engineering.

The research on tissue engineering applications has been progressing to manufacture ideal tissue scaffold biomaterials. In this study, a double-layered electrospun biofiber scaffold biomaterial including Polycaprolactone (PCL)/Collagen (COL) fibrous inner layer and PCL/ Momordica charantia (MC) and Hypericum perforatum (HP) oils fibrous outer layer was developed to manufacture a functional, novel tissue scaffold with the advantageous mechanical and biological properties. The main approach was to combine the natural perspective using medicinal oils with an engineering point of view to fabricate a potential functional scaffold for tissue engineering. Medicinal plants MC and HP are rich in functional oils and incorporation of them in a tissue scaffold will unveil their potential to augment both new tissue formation and wound healing. In this study, a novel double-layered scaffold prototype was fabricated using electrospinning technique with two PCL fiber layers, first is composed of collagen, and second is composed of oils extracted from medicinal plants. Initially, the composition of plant oils was analyzed. Thereafter the biofiber scaffold layers were fabricated and were evaluated in terms of morphology, physicochemistry, thermal and mechanical features, wettability, in vitro bio-degradability. Double-layered scaffold prototype was further analyzed in terms of in vitro biocompatibility and antibacterial effect. The medicinal oils blend provided antioxidant and antibacterial properties to the novel PCL/Oils layer. The results signify that inner PCL/COL layer exhibited advanced biodegradability of 8.5% compared to PCL and enhanced wettability with 11.7° contact angle. Strength of scaffold prototype was 5.98 N/mm2 thanks to the elastic PCL fibrous matrix. The double-layered functional biofiber scaffold enabled 92% viability after 72 h contact with fibroblast cells and furthermore provided feasible attachment sites for the cells. The functional scaffold prototype's noteworthy mechanical, chemical, and biological features enable it to be suggested as a different novel biomaterial with the potential to be utilized in tissue engineering applications.

Natural medicine delivery from 3D printed bone substitutes.

Unmet medical needs in treating critical-size bone defects have led to the development of numerous innovative bone tissue engineering implants. Although additive manufacturing allows flexible patient-specific treatments by modifying topological properties with various materials, the development of ideal bone implants that aid new tissue regeneration and reduce post-implantation bone disorders has been limited. Natural biomolecules are gaining the attention of the health industry due to their excellent safety profiles, providing equivalent or superior performances when compared to more expensive growth factors and synthetic drugs. Supplementing additive manufacturing with natural biomolecules enables the design of novel multifunctional bone implants that provide controlled biochemical delivery for bone tissue engineering applications. Controlled release of naturally derived biomolecules from a three-dimensional (3D) printed implant may improve implant-host tissue integration, new bone formation, bone healing, and blood vessel growth. The present review introduces us to the current progress and limitations of 3D printed bone implants with drug delivery capabilities, followed by an in-depth discussion on cutting-edge technologies for incorporating natural medicinal compounds embedded within the 3D printed scaffolds or on implant surfaces, highlighting their applications in several pre- and post-implantation bone-related disorders.

Porous scaffold hydroxyapatite from sand lobster shells (Panulirus homarus) using polyethylene oxide/chitosan as polymeric porogen for bone tissue engineering.

The hydroxyapatite (HAp; Ca10 (PO4 )6 (OH)2 )) has good biocompatibility, bioactivity, and osteoconductivity as a bone implant because the main inorganic mineral of human bone is HAp. The use of scaffold HAp from biogenic resources that contain high calcium and polymer as a pore forming agent to support bone growth is a longstanding area of interest. In this study, porous scaffolds based on HAp were synthesized from sand lobster (SL; Panulirus homarus) shells as a source of calcium using the porogen leaching method with polyethylene oxide (PEO) and chitosan (Chs) as polymeric porogen. The present study aims to synthesize HAp derived from SL shells and evaluate the effect variations of PEO on the physicochemical properties of the scaffold and cytotoxicity in cell viability assay. Briefly, the SL shell powder was calcinated with temperature variations of 600°C, 800°C, and 1000°C for 6 h. Based on the characterization, it was shown that 1000°C was the optimum calcination temperature for SL shells to synthesize HAp using the precipitation method. The characterization results of HAp using energy dispersive x-ray (EDX) revealed that the molar ratio of Ca/P was 1.67. The Fourier transform infrared (FTIR) and x-ray diffractometer (XRD) spectral patterns indicated that HAp had been successfully synthesized with minor ß-tricalcium phosphate (ß-TCP), a calcium phosphate with high biocompatibility. Porous scaffolds were synthesized by varying the concentration of PEO at 0, 5, 10, and 15 wt %. Physicochemical analysis revealed that a higher concentration of PEO affected decreased crystallinity and compressive strength, but on the other hand, the porosity and pore sizes increased. Based on the physicochemical analysis, the synthesized porous scaffold showed that HAp/PEO/Chs 15 wt % had the most potential as a scaffold for biomedical applications. MTT Assay, after 24 h incubation, revealed that the scaffold was safe for use at low concentrations on the MC3T3E1 osteoblast cells, with a percentage of cell viability of 83.23 ± 3.18% at 23.4375 µg/mL. Although the cell viability decreased at higher concentrations, the HAp/PEO/Chs 15 wt % scaffold was cytocompatible with the cells. Thus, in the present study, HAp/PEO/Chs 15 wt % was the best scaffold based on pore structure, chemical composition, mechanical and crystalographic properties and cell viability.

Biological Properties and Biomedical Applications of Pectin and Pectin-Based Composites: A Review.

Pectin has recently drawn much attention in biomedical applications due to its distinctive chemical and biological properties. Polymers like pectin with cell-instructive properties are attractive natural biomaterials for tissue repair and regeneration. In addition, bioactive pectin and pectin-based composites exhibit improved characteristics to deliver active molecules. Pectin and pectin-based composites serve as interactive matrices or scaffolds by stimulating cell adhesion and cell proliferation and enhancing tissue remodeling by forming an extracellular matrix in vivo. Several bioactive properties, such as immunoregulatory, antibacterial, anti-inflammatory, anti-tumor, and antioxidant activities, contribute to the pectin's and pectin-based composite's enhanced applications in tissue engineering and drug delivery systems. Tissue engineering scaffolds containing pectin and pectin-based conjugates or composites demonstrate essential features such as nontoxicity, tunable mechanical properties, biodegradability, and suitable surface properties. The design and fabrication of pectic composites are versatile for tissue engineering and drug delivery applications. This article reviews the promising characteristics of pectin or pectic polysaccharides and pectin-based composites and highlights their potential biomedical applications, focusing on drug delivery and tissue engineering.

Tea polyphenols coated sodium alginate-gelatin 3D edible scaffold for cultured meat.

This study aimed to use edible scaffolds as a platform for animal stem cell expansion, thus constructing block-shaped cell culture meat. The tea polyphenols (TP)-coated 3D scaffolds were constructed of sodium alginate (SA) and gelatin (Gel) with good biocompatibility and mechanical support. Initially, the physicochemical properties and mechanical properties of SA-Gel-TP scaffolds were measured, and the biocompatibility of the scaffolds was evaluated by C2C12 cells. SEM results showed that the scaffold had a porous laminar structure with TP particles attached to the surface, while FT-IR results also demonstrated the encapsulation of TP coating on the scaffold. In addition, the porosity of all scaffolds was higher than 40% and the degradation rate during the incubation cycle was less than 40% and the S2-G1-TP0.1-3 h scaffold has excellent cell adhesion and extension. Subsequently, we inoculated rabbit skeletal muscle myoblasts (RbSkMC) on the scaffold and induced differentiation. The results showed good adhesion and extension behavior of RbSkMC on S2-G1-TP0.1-3 h scaffolds with high expression of myogenic differentiation proteins and genes, and SEM results confirmed the formation of myotubes. Additionally, the adhesion rate of cells on scaffolds with TP coating was 1.5 times higher than that on scaffolds without coating, which significantly improved the cell proliferation rate and the morphology of cells with extension on the scaffolds. Furthermore, rabbit-derived cultured meat had similar appearance and textural characteristics to fresh meat. These conclusions indicate the high potential of the scaffolds with TP coating as a platform for the production of cultured meat products.

A Workflow to Produce a Low-Cost In Vitro Platform for the Electric-Field Pacing of Cellularised 3D Porous Scaffolds.

Endogenous electrically mediated signaling is a key feature of most native tissues, the most notable examples being the nervous and the cardiac systems. Biomedical engineering often aims to harness and drive such activity in vitro, in bioreactors to study cell disease and differentiation, and often in three-dimensional (3D) formats with the help of biomaterials, with most of these approaches adopting scaffold-free self-assembling strategies to create 3D tissues. In essence, this is the casting of gels which self-assemble in response to factors such as temperature or pH and have capacity to harbor cells during this process without imparting toxicity. However, the use of materials that do not self-assemble but can support 3D encapsulation of cells (such as porous scaffolds) warrants consideration given the larger repertoire this would provide in terms of material physicochemical properties and microstructure. In this method and protocol paper, we detail and provide design codes and assembly instructions to cheaply create an electrical pacing bioreactor and a Rig for Stimulation of Sponge-like Scaffolds (R3S). This setup has also been engineered to simultaneously perform live optical imaging of the in vitro models. To showcase a pilot exploration of material physiochemistry (in this aspect material conductivity) and microstructure (isotropy versus anisotropy), we adopt isotropic and anisotropic porous scaffolds composed of collagen or poly(3,4-ethylene dioxythiophene):polystyrenesulfonate (PEDOT:PSS) for their contrasting conductivity properties yet similar in porosity and mechanical integrity. Electric field pacing of mouse C3H10 cells on anisotropic porous scaffolds placed in R3S led to increased metabolic activity and enhanced cell alignment. Furthermore, after 7 days electrical pacing drove C3H10 alignment regardless of material conductivity or anisotropy. This platform and its design, which we have shared, have wide suitability for the study of electrical pacing of cellularized scaffolds in 3D in vitro cultures.

Selenium-Doped Mesoporous Bioactive Glass Regulates Macrophage Metabolism and Polarization by Scavenging ROS and Promotes Bone Regeneration In Vivo.

Bone regeneration is complex and involves multiple cells and systems, with macrophage-mediated immune regulation being critical for the development and regulation of inflammation, angiogenesis, and osteogenesis. Biomaterials with modified physical and chemical properties (e.g., modified wettability and morphology) effectively regulate macrophage polarization. This study proposes a novel approach to macrophage-polarization induction and -metabolism regulation through selenium (Se) doping. We synthesized Se-doped mesoporous bioactive glass (Se-MBG) and demonstrated its macrophage-polarization regulation toward M2 and its enhancement of the macrophage oxidative phosphorylation metabolism. The underlying mechanism is the effective scavenging of excessive intracellular reactive oxygen species (ROS) by the Se-MBG extracts through the promotion of peroxide-scavenging enzyme glutathione peroxidase 4 expression in the macrophages; this, in turn, improves the mitochondrial function. Printed Se-MBG scaffolds were implanted into rats with critical-sized skull defects to evaluate their immunomodulatory and bone regeneration capacity in vivo. The Se-MBG scaffolds demonstrated excellent immunomodulatory function and robust bone regeneration capacity. Macrophage depletion with clodronate liposomes impaired the Se-MBG-scaffold bone regeneration effect. Se-mediated immunomodulation, which targets ROS scavenging to regulate macrophage metabolic profiles and mitochondrial function, is a promising concept for future effective biomaterials for bone regeneration and immunomodulation.

3D printed Styrax Liquidus (Liquidambar orientalis Miller)-loaded poly (L-lactic acid)/chitosan based wound dressing material: Fabrication, characterization, and biocompatibility results.

The medicinal plant of Styrax liquidus (ST) (sweet gum balsam) which extracted from Liquidambar orientalis Mill tree, was loaded into the 3D printed polylactic acid (PLA)/chitosan (CS) based 3D printed scaffolds to investigate its wound healing and closure effect, in this study. The morphological and chemical properties of the ST loaded 3D printed scaffolds with different concentrations (1 %, 2 %, and 3 % wt) were investigated by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared Spectroscopy (FT-IR), respectively. In addition, the mechanical and thermal properties of the materials were investigated by Tensile test and Differential Scanning Calorimetry (DSC), respectively. The antimicrobial activities of the ST loaded 3D printed scaffolds and their incubation media in the PBS (pH 7.4, at 37 °C for 24 h) were investigated on two Gram-positive and two Gram-negative standard pathogenic bacteria with the agar disc diffusion method. The colorimetric MTT assay was used to determine the cell viability of human fibroblast cells (CCD-1072Sk) incubated with free ST, ST loaded, and unloaded 3D printed scaffolds. The 1 % and 2 % (wt) ST loaded PLA/CS/ST 3D printed scaffolds showed an increase in the cell number. Annexin V/PI double stain assay was performed to test whether early or late apoptosis was induced in the PLA/CS/1 % ST and PLA/CS/2 % ST loaded groups and the results were consistent with the MTT assay. Furthermore, a wound healing assay was carried out to investigate the effect of ST loaded 3D printed scaffolds on wound healing in CCD-1072Sk cells. The highest wound closure compared to the control group was observed on cells treated with PLA/CS/1 % ST for 72 h. According to the results, novel biocompatible ST loaded 3D printed scaffolds with antimicrobial effect can be used as wound healing material for potential tissue engineering applications.

Collagen-alginate 3D microscaffolds for studying cellular migration.

Metastasis is one of the major causes for cancer mortality. Its early steps comprise of invasion of basement membrane and migration. Thus, it is hypothesized that a platform, that allows quantification and grading of migration capability of cells can potentially be used for predicting metastatic potential. Two-dimensional (2D) models have been rendered inadequate for modelling in-vivo microenvironment due to various reasons. To attenuate homogeneity observed in 2D, three-dimensional (3D) platforms supplemented with bioinspired components have been designed. Unfortunately, till date there are no simple models to capture the migration of cells in 3D along with quantification of the process. In this study, we report an alginate-collagen based 3D model system, which can predict the migratory property of the cells within 72 h. The micron size of the scaffold enabled faster readout and the optimum pore-size provided conducive cellular growth environment. The platform's ability to allow observation of cellular migration was validated by encapsulating cells with transiently upregulated matrix metalloprotease 9 (MMP9), which has been reported to play a significant role in migration of cells during metastasis. The readout for migration was clustering of cells in the microscaffolds detected in a short span of 48 h. The observed clustering in MMP9 upregulated cells was validated by observing changes in the epithelial-mesenchymal transition (EMT) markers. Thus, this simple 3D platform can be used to study migration and predict the metastatic potential of cells.

A high molecular weight silk fibroin scaffold that resists degradation and promotes cell proliferation.

The regulation of the biodegradation rate of 3D-regenerated silk fibroin scaffolds and the avoidance of premature collapse are important concerns for their effective applications in tissue engineering. In this study, bromelain, which is specific to sericin, was used to remove sericin from silk, and high molecular weight silk fibroin was obtained after the fibroin fibers were dissolved. Afterwards, a 3D scaffold was prepared via freeze-drying. The Sodium dodecyl sulfate-polyacrylamide gel electrophoresis results showed that the average molecular weight of the regenerated silk fibroin prepared by using the bromelain-degumming method was approximately 142.2 kDa, which was significantly higher than that of the control groups prepared by using the urea- and Na2 CO3 -degumming methods. The results of enzyme degradation in vitro showed that the biodegradation rate and internal three-dimensional structure collapse of the bromelain-degumming fibroin scaffolds were significantly slower than those of the two control scaffolds. The proliferation activity of human umbilical vein vascular endothelial cells inoculated in bromelain-degumming fibroin scaffolds was significantly higher than that of the control scaffolds. This study provides a novel preparation method for 3D-regenerated silk fibroin scaffolds that can effectively resist biodegradation, continuously guide cell growth, have good biocompatibility, and have the potential to be used for the regeneration of various connective tissues.

Multifunctional Zn and Ag co-doped bioactive glass nanoparticles for bone therapeutic and regeneration.

Bone cancer has traditionally been treated using surgery, radiotherapy, and/or chemotherapy. The nonspecific distribution of chemotherapy and implantable infections are significant risk factors for the failure of the bone to heal. Multifunctional zinc and silver co-doped bioactive glass nanoparticles (yAg-xZn-BGNPs) with a diameter of 150 ± 30 nm were successfully synthesized using modified sol-gel and two-step post-functionalization processes, tailored to provide antibacterial and anticancer activity whilst maintaining osteogenesis ability. Co-doped BGNPs with Zn and Ag did not significantly alter physicochemical properties, including size, morphology, glass network, and amorphous nature. Apatite-like layer was observed on the surface of yAg-xZn-BGNPs and resorbed in the simulated body fluid solution, which could increase their bioactivity. Human fetal osteoblast cell line (hFOB 1.19) treated with particles showed calcified tissue formation and alkaline phosphatase activity in the absence of osteogenic supplements in vitro, especially with 0.5Ag-1Zn-BGNPs. Moreover, these particles preferentially disrupted the metabolic activity of bone cancer cells (MG-63) and had an antibacterial effect against B. subtilis, E. coli, and S. aureus via the disc diffusion method. This novel 0.5Ag-1Zn-BGNP and 1Ag-1Zn-BGNPs, with wide-ranging ability to stimulate bone regeneration, to inhibit bone cancer cell proliferation, and to prevent bacterial growth properties, may provide a feasible strategy for bone cancer treatment. The 0.5Ag-1Zn-BGNPs and 1Ag-1Zn-BGNPs can be applied for the preparation of scaffolds or filler composites using in bone tissue engineering.

Internally crosslinked alginate-based bioinks for the fabrication ofin vitrohepatic tissue models.

Bioprinting is a key technique to fabricate cell-laden volumetric constructs with controlled geometry. It can be used not only to replicate the architecture of a target organ but also to produce shapes that allow for the mimicry,in vitro,of specific desired features. Among the various materials suitable to be processed with this technique, sodium alginate is currently considered one of the most appealing because of its versatility. To date, the most widespread strategies to print alginate-based bioinks exploit external gelation as a primary process, by directly extruding the hydrogel-precursor solution into a crosslinking bath or within a sacrificial crosslinking hydrogel, where the gelation takes place. In this work, we describe the print optimization and the processing of Hep3Gel: an internally crosslinked alginate and ECM-based bioink for the production of volumetric hepatic tissue models. We adopted an unconventional strategy, by moving from the reproduction of the geometry and the architecture of liver tissue to the use of bioprinting to fabricate structures that can promote a high degree of oxygenation, as is the case with hepatic tissue. To this end, the design of structures was optimized by employing computational methods. The printability of the bioink was then studied and optimized through a combination of differenta priorianda posteriorianalyses. We produced 14-layered constructs, thus highlighting the possibility to exploit internal gelation alone to directly print self-standing structures with finely controlled viscoelastic properties. Constructs loaded with HepG2 cells were successfully printed and cultured in static conditions for up to 12 d, underlining the suitability of Hep3Gel to support mid/long-term cultures.

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.

Composite Graded Melt Electrowritten Scaffolds for Regeneration of the Periodontal Ligament-to-Bone Interface.

Periodontitis is a ubiquitous chronic inflammatory, bacteria-triggered oral disease affecting the adult population. If left untreated, periodontitis can lead to severe tissue destruction, eventually resulting in tooth loss. Despite previous efforts in clinically managing the disease, therapeutic strategies are still lacking. Herein, melt electrowriting (MEW) is utilized to develop a compositionally and structurally tailored graded scaffold for regeneration of the periodontal ligament-to-bone interface. The composite scaffolds, consisting of fibers of polycaprolactone (PCL) and fibers of PCL-containing magnesium phosphate (MgP) were fabricated using MEW. To maximize the bond between bone (MgP) and ligament (PCL) regions, we evaluated two different fiber architectures in the interface area. These were a crosshatch pattern at a 0/90° angle and a random pattern. MgP fibrous scaffolds were able to promote in vitro bone formation even in culture media devoid of osteogenic supplements. Mechanical properties after MgP incorporation resulted in an increase of the elastic modulus and yield stress of the scaffolds, and fiber orientation in the interfacial zone affected the interfacial toughness. Composite graded MEW scaffolds enhanced bone fill when they were implanted in an in vivo periodontal fenestration defect model in rats. The presence of an interfacial zone allows coordinated regeneration of multitissues, as indicated by higher expression of bone, ligament, and cementoblastic markers compared to empty defects. Collectively, MEW-fabricated scaffolds having compositionally and structurally tailored zones exhibit a good mimicry of the periodontal complex, with excellent regenerative capacity and great potential as a defect-specific treatment strategy.