Biomaterials-aided mandibular reconstruction using in vivo bioreactors.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.

Evaluation of tissue integration of injectable, cell-laden hydrogels of cocultures of mesenchymal stem cells and articular chondrocytes with an ex vivo cartilage explant model.

This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N-isopropylacrylamide)-based dual-network hydrogels loaded with poly( l -lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels-specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC-encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties.

Influence of Polymeric Microparticle Size and Loading Concentration on 3D Printing Accuracy and Degradation Behavior of Composite Scaffolds.

Successful employment of 3D printing for delivery of therapeutic biomolecules requires protection of their bioactivity on exposure to potentially inactivating conditions. Although intermediary encapsulation of the biomolecules in polymeric particulate delivery vehicles is a promising strategy for this objective, the inclusion of such particles in 3D printing formulations may critically impact the accuracy or precision of 3D printed scaffolds relative to their intended designed architectures, as well as the degradation behavior of both the scaffolds and the included particles. The present work aimed to elucidate the effect of poly(d,l-lactic-co-glycolic acid) particle size and loading concentration on material accuracy, machine precision, and degradation of 3D printed poly(ɛ-caprolactone)-based scaffolds. Using a main effects analysis, the sizes and loading concentrations of particle delivery vehicles investigated were found to have neither a beneficial nor disadvantageous influence on the metrics of printing quality such as material accuracy and machine precision. Meanwhile, particle loading concentration was determined to influence degradation rate, whereas printing temperature affected the trends in composite weight-average molecular weight. Neither of the two particle-related parameters (concentration nor diameter) was found to exhibit a significant effect on intra-fiber nor inter-fiber porosity. These findings evidence the capacity for controlled loading of particulate delivery vehicles in 3D printed scaffolds while preserving construct accuracy and precision, and with predictable dictation of composite degradation behavior for potential controlled release of encapsulated biomolecules.

Bioinspired electrospun decellularized extracellular matrix scaffolds promote muscle regeneration in a rat skeletal muscle defect model.

Volumetric muscle loss is a debilitating injury that can leave patients with long-lasting or permanent structural and functional deficits. With clinical treatments failing to address these shortcomings, there is a great need for tissue-engineered therapies to promote skeletal muscle regeneration. In this study, we aim to assess the potential for electrospun decellularized skeletal muscle extracellular matrix (dECM) to promote skeletal muscle regeneration in a rat partial thickness tibialis anterior defect model. Aligned electrospun scaffolds with varying degrees of crosslinking density were implanted into the defect site and compared to an empty defect control. After 8 weeks, muscles were harvested, weighed, and cellular and morphological analyses were performed via histology and immunohistochemistry. Cell infiltration, angiogenesis, and myogenesis were observed in the defect site in both dECM groups. However, favorable mechanical properties and slower degradation kinetics resulted in greater support of tissue remodeling in the more crosslinked scaffolds and preservation of existing myofiber area in both dECM groups compared to the empty defect control. More sustained release of pro-regenerative degradation products also promoted greater myofiber formation in the defect site. This study allowed for a greater understanding of how electrospun skeletal muscle scaffolds interact with existing skeletal muscle and can inform their potential as a therapy in a wide variety of soft tissue applications.

3D printed colloidal biomaterials based on photo-reactive gelatin nanoparticles.

Biomaterials-based strategies have shown great promise for tissue regeneration. 3D printing technologies can deliver unprecedented control over architecture and properties of biomaterial constructs when combined with innovative material design strategies. Colloidal gels made of polymeric nanoparticles are attractive injectable and self-healing systems, but their use as bio-inks for extrusion-based printing is largely unexplored. Here, we report 3D printing of novel biomaterial constructs with shape memory behavior using photo-reactive gelatin nanoparticles as colloidal building blocks. These nanoparticles are stabilized with intraparticle covalent crosslinks, and also contain pendant methacryloyl groups as photo-reactive moieties. While non-covalent interactions between nanoparticles enable formation of colloidal gel inks that are printable at room temperature, UV-induced covalent interparticle crosslinks based on methacryloyl moieties significantly enhance mechanical properties of printed constructs. Additionally, the UV crosslinking modality enables remarkable control over swelling, degradation, and biomolecule release behavior of 3D constructs. Finally, by exploiting the mechanical properties of colloidal biomaterials after UV crosslinking, 3D constructs can be designed with shape memory properties, returning to their original programmed geometry upon re-hydration. Accordingly, these novel colloidal inks exhibit great potential to serve as bio-inks for 3D printing of biomaterials with shape-morphing features for a wide range of tissue engineering and regenerative medicine applications.

Effect of 3D Printing Temperature on Bioactivity of Bone Morphogenetic Protein-2 Released from Polymeric Constructs.

Growth factors such as bone morphogenetic protein-2 (BMP-2) are potent tools for tissue engineering. Three-dimensional (3D) printing offers a potential strategy for delivery of BMP-2 from polymeric constructs; however, these biomolecules are sensitive to inactivation by the elevated temperatures commonly employed during extrusion-based 3D printing. Therefore, we aimed to correlate printing temperature to the bioactivity of BMP-2 released from 3D printed constructs composed of a model polymer, poly(propylene fumarate). Following encapsulation of BMP-2 in poly(DL-lactic-co-glycolic acid) particles, growth factor-loaded fibers were fabricated at three different printing temperatures. Resulting constructs underwent 28 days of aqueous degradation for collection of released BMP-2. Supernatants were then assayed for the presence of bioactive BMP-2 using a cellular assay for alkaline phosphatase activity. Cumulative release profiles indicated that BMP-2 released from constructs that were 3D printed at physiologic and intermediate temperatures exhibited comparable total amounts of bioactive BMP-2 release as those encapsulated in non-printed particulate delivery vehicles. Meanwhile, the elevated printing temperature of 90 °C resulted in a decreased amount of total bioactive BMP-2 release from the fibers. These findings elucidate the effects of elevated printing temperatures on BMP-2 bioactivity during extrusion-based 3D printing, and enlighten polymeric material selection for 3D printing with growth factors.

Bilayered, peptide-biofunctionalized hydrogels for in vivo osteochondral tissue repair.

Osteochondral defects present a unique clinical challenge due to their combination of phenotypically distinct cartilage and bone, which require specific, stratified biochemical cues for tissue regeneration. Furthermore, the articular cartilage exhibits significantly worse regeneration than bone due to its largely acellular and avascular nature, prompting significant demand for regenerative therapies. To address these clinical challenges, we have developed a bilayered, modular hydrogel system that enables the click functionalization of cartilage- and bone-specific biochemical cues to each layer. In this system, the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) was click conjugated with either a cartilage- or bone-specific peptide sequence of interest, and then mixed with a suspension of thermoresponsive polymer and mesenchymal stem cells (MSCs) to generate tissue-specific, cell-encapsulated hydrogel layers targeting the cartilage or bone. We implanted bilayered hydrogels in rabbit femoral condyle defects and investigated the effects of tissue-specific peptide presentation and cell encapsulation on osteochondral tissue repair. After 12 weeks implantation, hydrogels with a chondrogenic peptide sequence produced higher histological measures of overall defect filling, cartilage surface regularity, glycosaminoglycan (GAG)/cell content of neocartilage and adjacent cartilage, and bone filling and bonding compared to non-chondrogenic hydrogels. Furthermore, MSC encapsulation promoted greater histological measures of overall defect filling, cartilage thickness, GAG/cell content of neocartilage, and bone filling. Our results establish the utility of this click functionalized hydrogel system for in vivo repair of the osteochondral unit. STATEMENT OF SIGNIFICANCE: Osteochondral repair requires mimicry of both cartilage- and bone-specific biochemical cues, which are highly distinct. While traditional constructs for osteochondral repair have mimicked gross compositional differences between the cartilage and bone in mineral content, mechanical properties, proteins, or cell types, few constructs have recapitulated the specific biochemical cues responsible for the differential development of cartilage and bone. In this study, click biofunctionalized, bilayered hydrogels produced stratified presentation of developmentally inspired peptide sequences for chondrogenesis and osteogenesis. This work represents, to the authors' knowledge, the first application of bioconjugation chemistry for the simultaneous repair of bone and cartilage tissue. The conjugation of tissue-specific peptide sequences successfully promoted development of both cartilage and bone tissues in vivo.

Tuning pore features of mineralized collagen/PCL scaffolds for cranial bone regeneration in a rat model.

Porosity is indispensable for a bone tissue-engineered scaffold for facilitating endogenous cell migration and nascent bone ingrowth. In large-sized cranial bone defect repair, porous scaffolds meet great challenges to match cranial bone regeneration and provide sufficient protection with structural integrity. Therefore, the pore features of the scaffolds for cranial bone regeneration should differ from those typical porous scaffolds used in tubular bone repair and be finely tuned. In this study, a series of porous mineralized collagen/PCL scaffolds with different pore features were fabricated via freeze-drying and applied in a Sprague Dawley rat cranial bone calvarial defect model. The pore size for four groups increased from 10-45 µm to 40-130 µm. As scaffold porosity increased, the compressive strength decreased from 2.09 ±â€¯0.12 MPa to 0.51 ±â€¯0.04 MPa. The micro-computed tomography three-dimensional reconstruction images showed that as pore size and porosity increased, the amount of new bone formation had a maximum in group 3 (pore size: 20-100 µm, compressive strength: 1.06 ±â€¯0.03 MPa). In addition, the histological and histomorphometric analyses showed a consistent tendency which confirmed the Micro-CT results. Meanwhile, histological findings including bony bridging, tissue response at the bone-implant interface and fibrous capsule thickness indicated that the dura mater pathway played the most important role in the regenerative process of this calvarial defect model.

Fiber engraving for bioink bioprinting within 3D printed tissue engineering scaffolds.

In this work, we describe a new 3D printing methodology for the fabrication of multimaterial scaffolds involving the combination of thermoplastic extrusion and low temperature extrusion of bioinks. A fiber engraving technique was used to create a groove on the surface of a thermoplastic printed fiber using a commercial 3D printer and a low viscosity bioink was deposited into this groove. In contrast to traditional extrusion bioinks that rely on increased viscosity to prevent lateral spreading, this groove creates a defined space for bioink deposition. By physically constraining bioink spreading, a broader range of viscosities can be used. As proof-of-concept, we fabricated and characterized a multimaterial scaffold containing poly(ε-caprolactone) (PCL) as the thermoplastic polymer and a gelatin-based bioink. A 7.5 w/v% gelatin methacryloyl (GelMA) bioink loaded with either 5 w/v% poly(lactic-co-glycolic acid) (PLGA) microparticles containing fluorescent albumin or mouse fibroblasts (1 × 106 cell/mL) was printed at 24 °C. The structure of the composite scaffolds had no significant decrease in porosity or mechanical properties as compared to the PCL control scaffolds, demonstrating the engraving technique did not significantly compromise the mechanical or structural integrity of the scaffold. The encapsulated PLGA microparticles were homogeneously distributed in the GelMA and remained in the scaffolds after incubation in PBS for 24 h at 37 °C. In addition, the viability of the fibroblasts encapsulated in the GelMA bioink and printed in the grooves of the PCL scaffolds was confirmed after 24 h of incubation. Overall, this work provides a new methodology for the preparation of 3D printed scaffolds containing a robust thermoplastic structure in combination with low viscosity bioinks.

Three-Dimensional Extrusion Printing of Porous Scaffolds Using Storable Ceramic Inks.

In this study, we describe the additive manufacturing of porous three-dimensionally (3D) printed ceramic scaffolds prepared with hydroxyapatite (HA), ß-tricalcium phosphate (ß-TCP), or the combination of both with an extrusion-based process. The scaffolds were printed using a novel ceramic-based ink with reproducible printability and storability properties. After sintering at 1200°C, the scaffolds were characterized in terms of structure, mechanical properties, and dissolution in aqueous medium. Microcomputed tomography and scanning electron microscopy analyses revealed that the structure of the scaffolds, and more specifically, pore size, porosity, and isotropic dimensions were not significantly affected by the sintering process, resulting in scaffolds that closely replicate the original dimensions of the 3D model design. The mechanical properties of the sintered scaffolds were in the range of human trabecular bone for all compositions. All ceramic bioinks showed consistent printability over a span of 14 days, demonstrating the short-term storability of the formulations. Finally, the mass loss did not vary among the evaluated compositions over a period of 28 days except in the case of ß-TCP scaffolds, in which the structural integrity was significantly affected after 28 days of incubation in phosphate-buffered saline. In conclusion, this study demonstrates the development of storable ceramic inks for the 3D printing of scaffolds of HA, ß-TCP, and mixtures thereof with high fidelity and low shrinkage following sintering that could potentially be used for bone tissue engineering in load-bearing applications.

Chondrogenesis of cocultures of mesenchymal stem cells and articular chondrocytes in poly(l-lysine)-loaded hydrogels.

This work investigated the effect of poly(l-lysine) (PLL) molecular weight and concentration on chondrogenesis of cocultures of mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) in PLL-loaded hydrogels. An injectable dual-network hydrogel composed of a poly(N-isopropylacrylamide)-based synthetic thermogelling macromer and a chondroitin sulfate-based biological network was leveraged as a model to deliver PLL and encapsulate the two cell populations. Incorporation of PLL into the hydrogel did not affect the hydrogel's swelling properties and degradation characteristics, nor the viability of encapsulated cells. Coculture groups demonstrated higher type II collagen expression compared to the MSC monoculture group. Expression of hypertrophic phenotype was also limited in the coculture groups. Histological analysis indicated that the ratio of MSCs to ACs was an accurate predictor of the degree of long-term chondrogenesis, while the presence of PLL was shown to have a more substantial short-term effect. Altogether, this study demonstrates that coculturing MSCs with ACs can greatly enhance the chondrogenicity of the overall cell population and offers a platform to further elucidate the short- and long-term effect of polycationic factors on the chondrogenesis of MSC and AC cocultures.

3D Tissue-Engineered Tumor Model for Ewing's Sarcoma That Incorporates Bone-like ECM and Mineralization.

The tumor microenvironment harbors essential components required for cancer progression including biochemical signals and mechanical cues. To study the effects of microenvironmental elements on Ewing's sarcoma (ES) pathogenesis, we tissue-engineered an acellular three-dimensional (3D) bone tumor niche from electrospun poly(ε-caprolactone) (PCL) scaffolds that incorporate bone-like architecture, extracellular matrix (ECM), and mineralization. PCL-ECM constructs were generated by decellularizing PCL scaffolds harboring cultures of osteogenic human mesenchymal stem cells. The PCL-ECM constructs simulated in vivo-like tumor architecture and increased the proliferation of ES cells compared to PCL scaffolds alone. Compared to monolayer controls, 3D environments facilitated the downregulation of the canonical insulin-like growth factor 1 receptor (IGF-1R) signal cascade through mechanistic target of rapamycin (mTOR), both of which are targets of recent clinical trials. In addition to the downregulation of canonical IGF-1R signaling, 3D environments promoted a reduction in the clathrin-dependent nuclear localization and transcriptional activity of IGF-1R. In vitro drug testing revealed that 3D environments generated cell phenotypes that were resistant to mTOR inhibition and chemotherapy. Our versatile PCL-ECM constructs allow for the investigation of the roles of various microenvironmental elements in ES tumor growth, cancer cell morphology, and induction of resistant cell phenotypes.

Progress in three-dimensional printing with growth factors.

Incorporation of growth factors in biomedical constructs can encourage cellular activities necessary for tissue regeneration within an implant system. Three-dimensional printing offers a capacity for spatial dictation and dosage control of incorporated growth factors which promises to minimize complications from the supraphysiologic doses and burst release involved in current growth factor delivery systems. Successful implementation of three-dimensional printing with growth factors requires preservation of the bioactivity of printed growth factors, spatial localization of growth factors within the construct architecture during printing, and controlled release of growth factors after printing. This review describes demonstrated approaches for addressing each of these goals, including direct inclusion of growth factors with the biomaterial during printing, or intermediary encapsulation of growth factors in delivery vehicles such as microparticles or nanoparticles.

Why, When, Who, What, How, and Where for Trainees Writing Literature Review Articles.

Literature review articles provide a valuable mechanism for remaining informed amidst an ever-increasing body of scientific work. Condensing current advances into this disseminatable form is a critical activity for any research trainee. To systematize this multifaceted process, we present the "why, when, who, what, how, and where" of composing a literature review article. Commentaries include selection of a review topic, conducting modern literature searches using online databases, stepwise strategies for manuscript drafting, and prevention of plagiarism. The current work provides structures and guidance for this fundamental effort which can establish the basis for a trainee's development of original research objectives.

Insect Bite-Associated Invasive Fungal Infections.

Insect bites are rarely reported to result in myocutaneous mycoses. We reviewed the literature and report 22 cases. Molds were the most common pathogens (15), especially Mucorales (9). Infections were typically misdiagnosed, and 68% had necrotizing features, often requiring amputation or extensive surgery. Both immunocompetent and immunosuppressed patients were affected.

A review on the exploitation of biodegradable magnesium-based composites for medical applications.

In recent years, materials science research based on magnesium (Mg) alloys has increased significantly due to their notable advantages over traditional metals. However, magnesium alloys are susceptible to excessive degradation and subsequent disruption of mechanical integrity; this phenomenon limits the utility of these materials. Mg alloys can thus be combined with other materials to form composites for medical applications. The present article describes key advances in and considerations for the development of biodegradable Mg-based composites (BMCs). The primary characteristics of these materials include their controllable degradation rates, tunable mechanical properties, adjustable structures to promote tissue repair, improved biocompatibility, and added functionality according to the purpose of the applications. Here we provide an overview of the current research on and development status of BMCs for biomedical materials, including the present limitations and challenges of their use. Finally, this paper comprehensively discusses the most promising directions of future development for these materials.