Advances in 3D bioprinting for regenerative medicine applications.

Biofabrication techniques allow for the construction of biocompatible and biofunctional structures composed from biomaterials, cells and biomolecules. Bioprinting is an emerging 3D printing method which utilizes biomaterial-based mixtures with cells and other biological constituents into printable suspensions known as bioinks. Coupled with automated design protocols and based on different modes for droplet deposition, 3D bioprinters are able to fabricate hydrogel-based objects with specific architecture and geometrical properties, providing the necessary environment that promotes cell growth and directs cell differentiation towards application-related lineages. For the preparation of such bioinks, various water-soluble biomaterials have been employed, including natural and synthetic biopolymers, and inorganic materials. Bioprinted constructs are considered to be one of the most promising avenues in regenerative medicine due to their native organ biomimicry. For a successful application, the bioprinted constructs should meet particular criteria such as optimal biological response, mechanical properties similar to the target tissue, high levels of reproducibility and printing fidelity, but also increased upscaling capability. In this review, we highlight the most recent advances in bioprinting, focusing on the regeneration of various tissues including bone, cartilage, cardiovascular, neural, skin and other organs such as liver, kidney, pancreas and lungs. We discuss the rapidly developing co-culture bioprinting systems used to resemble the complexity of tissues and organs and the crosstalk between various cell populations towards regeneration. Moreover, we report on the basic physical principles governing 3D bioprinting, and the ideal bioink properties based on the biomaterials' regenerative potential. We examine and critically discuss the present status of 3D bioprinting regarding its applicability and current limitations that need to be overcome to establish it at the forefront of artificial organ production and transplantation.

Mapping the Single-Cell Differentiation Landscape of Osteosarcoma.

PURPOSE: The genetic intratumoral heterogeneity observed in human osteosarcomas poses challenges for drug development and the study of cell fate, plasticity, and differentiation, which are processes linked to tumor grade, cell metastasis, and survival. EXPERIMENTAL DESIGN: To pinpoint errors in osteosarcoma differentiation, we transcriptionally profiled 31,527 cells from a tissue-engineered model that directs mesenchymal stem cells toward adipogenic and osteoblastic fates. Incorporating preexisting chondrocyte data, we applied trajectory analysis and non-negative matrix factorization to generate the first human mesenchymal differentiation atlas. RESULTS: This "roadmap" served as a reference to delineate the cellular composition of morphologically complex osteosarcoma tumors and quantify each cell's lineage commitment. Projecting a bulk RNA-sequencing osteosarcoma dataset onto this roadmap unveiled a correlation between a stem-like transcriptomic phenotype and poorer survival outcomes. CONCLUSIONS: Our study quantifies osteosarcoma differentiation and lineage, a prerequisite to better understanding lineage-specific differentiation bottlenecks that might someday be targeted therapeutically.

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.

Bone mimetic environments support engineering, propagation, and analysis of therapeutic response of patient-derived cells, ex vivo and in vivo.

Bone metastases are the most common milestone in the lethal progression of prostate cancer and prominent in a substantial portion of renal malignancies. Interactions between cancer and bone host cells have emerged as drivers of both disease progression and therapeutic resistance. To best understand these central host-epithelial cell interactions, biologically relevant preclinical models are required. To achieve this goal, we here established and characterized tissue-engineered bone mimetic environments (BME) capable of supporting the growth of patient-derived xenograft (PDX) cells, ex vivo and in vivo. The BME consisted of a polycaprolactone (PCL) scaffold colonized by human mesenchymal stem cells (hMSCs) differentiated into osteoblasts. PDX-derived cells were isolated from bone metastatic prostate or renal tumors, engineered to express GFP or luciferase and seeded onto the BMEs. BMEs supported the growth and therapy response of PDX-derived cells, ex vivo. Additionally, BMEs survived after in vivo implantation and further sustained the growth of PDX-derived cells, their serial transplant, and their application to study the response to treatment. Taken together, this demonstrates the utility of BMEs in combination with patient-derived cells, both ex vivo and in vivo. STATEMENT OF SIGNIFICANCE: Our tissue-engineered BME supported the growth of patient-derived cells and proved useful to monitor the therapy response, both ex vivo and in vivo. This approach has the potential to enable co-clinical strategies to monitor bone metastatic tumor progression and therapy response, including identification and prioritization of new targets for patient treatment.

Targeting prostate tumor low-molecular weight tyrosine phosphatase for oxidation-sensitizing therapy.

Protein tyrosine phosphatases (PTPs) play major roles in cancer and are emerging as therapeutic targets. Recent reports suggest low-molecular weight PTP (LMPTP)-encoded by the ACP1 gene-is overexpressed in prostate tumors. We found ACP1 up-regulated in human prostate tumors and ACP1 expression inversely correlated with overall survival. Using CRISPR-Cas9-generated LMPTP knockout C4-2B and MyC-CaP cells, we identified LMPTP as a critical promoter of prostate cancer (PCa) growth and bone metastasis. Through metabolomics, we found that LMPTP promotes PCa cell glutathione synthesis by dephosphorylating glutathione synthetase on inhibitory Tyr270. PCa cells lacking LMPTP showed reduced glutathione, enhanced activation of eukaryotic initiation factor 2-mediated stress response, and enhanced reactive oxygen species after exposure to taxane drugs. LMPTP inhibition slowed primary and bone metastatic prostate tumor growth in mice. These findings reveal a role for LMPTP as a critical promoter of PCa growth and metastasis and validate LMPTP inhibition as a therapeutic strategy for treating PCa through sensitization to oxidative stress.

Self-rectifying magnetoelectric metamaterials for remote neural stimulation and motor function restoration.

Magnetoelectric materials convert magnetic fields into electric fields. These materials are often used in wireless electronic and biomedical applications. For example, magnetoelectrics could enable the remote stimulation of neural tissue, but the optimal resonance frequencies are typically too high to stimulate neural activity. Here we describe a self-rectifying magnetoelectric metamaterial for a precisely timed neural stimulation. This metamaterial relies on nonlinear charge transport across semiconductor layers that allow the material to generate a steady bias voltage in the presence of an alternating magnetic field. We generate arbitrary pulse sequences with time-averaged voltage biases in excess of 2 V. As a result, we can use magnetoelectric nonlinear metamaterials to wirelessly stimulate peripheral nerves to restore a sensory reflex in an anaesthetized rat model and restore signal propagation in a severed nerve with latencies of less than 5 ms. Overall, these results showing the rational design of magnetoelectric metamaterials support applications in advanced biotechnology and electronics.

Development of photoreactive demineralized bone matrix 3D printing colloidal inks for bone tissue engineering.

Demineralized bone matrix (DBM) has been widely used clinically for dental, craniofacial and skeletal bone repair, as an osteoinductive and osteoconductive material. 3D printing (3DP) enables the creation of bone tissue engineering scaffolds with complex geometries and porosity. Photoreactive methacryloylated gelatin nanoparticles (GNP-MAs) 3DP inks have been developed, which display gel-like behavior for high print fidelity and are capable of post-printing photocrosslinking for control of scaffold swelling and degradation. Here, novel DBM nanoparticles (DBM-NPs, ∼400 nm) were fabricated and characterized prior to incorporation in 3DP inks. The objectives of this study were to determine how these DBM-NPs would influence the printability of composite colloidal 3DP inks, assess the impact of ultraviolet (UV) crosslinking on 3DP scaffold swelling and degradation and evaluate the osteogenic potential of DBM-NP-containing composite colloidal scaffolds. The addition of methacryloylated DBM-NPs (DBM-NP-MAs) to composite colloidal inks (100:0, 95:5 and 75:25 GNP-MA:DBM-NP-MA) did not significantly impact the rheological properties associated with printability, such as viscosity and shear recovery or photocrosslinking. UV crosslinking with a UV dosage of 3 J/cm2 directly impacted the rate of 3DP scaffold swelling for all GNP-MA:DBM-NP-MA ratios with an ∼40% greater increase in scaffold area and pore area in uncrosslinked versus photocrosslinked scaffolds over 21 days in phosphate-buffered saline (PBS). Likewise, degradation (hydrolytic and enzymatic) over 21 days for all DBM-NP-MA content groups was significantly decreased, ∼45% less in PBS and collagenase-containing PBS, in UV-crosslinked versus uncrosslinked groups. The incorporation of DBM-NP-MAs into scaffolds decreased mass loss compared to GNP-MA-only scaffolds during collagenase degradation. An in vitro osteogenic study with bone marrow-derived mesenchymal stem cells demonstrated osteoconductive properties of 3DP scaffolds for the DBM-NP-MA contents examined. The creation of photoreactive DBM-NP-MAs and their application in 3DP provide a platform for the development of ECM-derived colloidal materials and tailored control of biochemical cue presentation with broad tissue engineering applications.

Corrigendum to "Advances in In Vitro and In Vivo Bioreactor-Based Bone Generation for Craniofacial Tissue Engineering".

[This corrects the article DOI: 10.34133/bmef.0004.].

Advances in In Vitro and In Vivo Bioreactor-Based Bone Generation for Craniofacial Tissue Engineering.

Craniofacial reconstruction requires robust bone of specified geometry for the repair to be both functional and aesthetic. While native bone from elsewhere in the body can be harvested, shaped, and implanted within a defect, using either an in vitro or in vivo bioreactors eliminates donor site morbidity while increasing the customizability of the generated tissue. In vitro bioreactors utilize cells harvested from the patient, a scaffold, and a device to increase mass transfer of nutrients, oxygen, and waste, allowing for generation of larger viable tissues. In vivo bioreactors utilize the patient's own body as a source of cells and of nutrient transfer and involve the implantation of a scaffold with or without growth factors adjacent to vasculature, followed by the eventual transfer of vascularized, mineralized tissue to the defect site. Several different models of in vitro bioreactors exist, and several different implantation sites have been successfully utilized for in vivo tissue generation and defect repair in humans. In this review, we discuss the specifics of each bioreactor strategy, as well as the advantages and disadvantages of each and the future directions for the engineering of bony tissues for craniofacial defect repair.

Managing Temporomandibular Joint Osteoarthritis by Dental Stem Cell Secretome.

The potential therapeutic role of the Dental Pulp Stem Cells Secretome (SECR) in a rat model of experimentally induced Temporomandibular Joint (TMJ) Osteoarthritis (OA) was evaluated. Proteomic profiling of the human SECR under specific oxygen tension (5% O2) and stimulation with Tumor Necrosis Factor-alpha (TNF-α) was performed. SECR and respective cell lysates (CL) samples were collected and subjected to SDS-PAGE, followed by LC-MS/MS analysis. The identified proteins were analyzed with Bioinformatic tools. The anti-inflammatory properties of SECR were assessed via an in vitro murine macrophages model, and were further validated in vivo, in a rat model of chemically-induced TMJ-OA by weekly recording of the head withdrawal threshold, the food intake, and the weight change, and radiographically and histologically at 4- and 8-weeks post-treatment. SECR analysis revealed the presence of 50 proteins that were enriched and/or statistically significantly upregulated compared to CL, while many of those proteins were involved in pathways related to "extracellular matrix organization" and "immune system". SECR application in vitro led to a significant downregulation on the expression of pro-inflammatory genes (MMP-13, MMP-9, MMP-3 and MCP-1), while maintaining an increased expression of IL-10 and IL-6. SECR application in vivo had a significant positive effect on all the clinical parameters, resulting in improved food intake, weight, and pain suppression. Radiographically, SECR application had a significant positive effect on trabecular bone thickness and bone density compared to the saline-treated group. Histological analysis indicated that SECR administration reduced inflammation, enhanced ECM and subchondral bone repair and regeneration, thus alleviating TMJ degeneration.

Mapping the Single-cell Differentiation Landscape of Osteosarcoma.

The genetic and intratumoral heterogeneity observed in human osteosarcomas (OS) poses challenges for drug development and the study of cell fate, plasticity, and differentiation, processes linked to tumor grade, cell metastasis, and survival. To pinpoint errors in OS differentiation, we transcriptionally profiled 31,527 cells from a tissue-engineered model that directs MSCs toward adipogenic and osteoblastic fates. Incorporating pre-existing chondrocyte data, we applied trajectory analysis and non-negative matrix factorization (NMF) to generate the first human mesenchymal differentiation atlas. This 'roadmap' served as a reference to delineate the cellular composition of morphologically complex OS tumors and quantify each cell's lineage commitment. Projecting these signatures onto a bulk RNA-seq OS dataset unveiled a correlation between a stem-like transcriptomic phenotype and poorer survival outcomes. Our study takes the critical first step in accurately quantifying OS differentiation and lineage, a prerequisite to better understanding global differentiation bottlenecks that might someday be targeted therapeutically.

Extracellular matrix component-derived nanoparticles for drug delivery and tissue engineering.

The extracellular matrix (ECM) consists of a complex combination of proteins, proteoglycans, and other biomolecules. ECM-based materials have been demonstrated to have high biocompatibility and bioactivity, which may be harnessed for drug delivery and tissue engineering applications. Herein, nanoparticles incorporating ECM-based materials and their applications in drug delivery and tissue engineering are reviewed. Proteins such as gelatin, collagen, and fibrin as well as glycosaminoglycans including hyaluronic acid, chondroitin sulfate, and heparin have been employed for cancer therapeutic delivery, gene delivery, and wound healing and regenerative medicine. Strategies for modifying and functionalizing these materials with synthetic and natural polymers or to enable stimuli-responsive degradation and drug release have increased the efficacy of these materials and nano-systems. The incorporation and modification of ECM-based materials may be used to drive drug targeting and increase tissue-specific cell differentiation more effectively.

Aspects of a Suspended Bioprinting System Affect Cell Viability and Support Bath Properties.

Suspended hydrogel printing is a growing method for fabricating bioprinted hydrogel constructs, largely due to how it enables nonviscous hydrogel inks to be used in extrusion printing. In this work, a previously developed poly(N-isopropylacrylamide)-based thermogelling suspended bioprinting system was examined in the context of chondrocyte-laden printing. Material factors such as ink concentration and cell concentration were found to have a significant effect on printed chondrocyte viability. In addition, the heated poloxamer support bath was able to maintain chondrocyte viability for up to 6 h of residence within the bath. The relationship between the ink and support bath was also assessed by measuring the rheological properties of the bath before and after printing. Bath storage modulus and yield stress decreased during printing as nozzle size was reduced, indicating the likelihood that dilution occurs over time through osmotic exchange with the ink. Altogether this work demonstrates the promise for printing high-resolution cell-encapsulating tissue engineering constructs, while also elucidating complex relationships between the ink and bath, which must be taken into consideration when designing suspended printing systems.

Particle carriers for controlled release of peptides.

There has been growing discovery and use of therapeutic peptides in drug delivery and tissue engineering. Peptides are smaller than proteins and can be formulated into drug delivery systems without significant loss of their bioactivity, which remains a concern with proteins. However, the smaller size of peptides has made the controlled release of these bioactive molecules from carriers challenging. Thus, there has been increasing development of carriers to improve the controlled release of peptides by leveraging hydrophobic and electrostatic interactions between the peptide and the carrier. The focus of this review paper is to critically discuss synthetic and natural nanoparticles and microparticles that have been investigated for the controlled delivery of peptides with emphasis on the underlying interactions.

Lateral Bone Augmentation Using a Three-Dimensional-Printed Polymeric Chamber to Compare Biomaterials.

The aim of this study was to test the suitability of calcium phosphate cement mixed with poly(lactic-co-glycolic acid) (CPC-PLGA) microparticles into a ring-shaped polymeric space-maintaining device as bone graft material for lateral bone augmentation. Therefore, the bone chambers were installed on the lateral portion of the anterior region of the mandibular body of mini-pigs. Chambers were filled with either CPC-PLGA or BioOss® particles for comparison and left for 4 and 12 weeks. Histology and histomorphometry were used to obtain temporal insight in material degradation and bone formation. Results indicated that between 4 and 12 weeks of implantation, a significant degradation of the CPC-PLGA (from 75.1% to 23.1%), as well as BioOss material, occurred (from 40.6% to 14.4%). Degradation of both materials was associated with the presence of macrophage-like and osteoclast-like cells. Furthermore, a significant increase in bone formation occurred between 4 and 12 weeks for the CPC-PLGA (from 0.1% to 7.2%), as well as BioOss material (from 8.3% to 23.3%). Statistical analysis showed that bone formation had progressed significantly better using BioOss compared to CPC-PLGA (p < 0.05). In conclusion, this mini-pig study showed that CPC-PLGA does not stimulate lateral bone augmentation using a bone chamber device. Both treatments failed to achieve "clinically" meaningful alveolar ridge augmentation.

High Strength Titanium with Fibrous Grain for Advanced Bone Regeneration.

Pure titanium is widely used in clinical implants, but its bioinert properties (poor strength and mediocre effect on bone healing) limit its use under load-bearing conditions. Modeling on the structure of collagen fibrils and specific nanocrystal plane arrangement of hydroxyapatite in the natural bone, a new type of titanium (Ti) with a highly aligned fibrous-grained (FG) microstructure is constructed. The improved attributes of FG Ti include high strength (≈950 MPa), outstanding affinity to new bone growth, and tight bone-implant contact. The bone-mimicking fibrous grains induce an aligned surface topological structure conducive to forming close contact with osteoblasts and promotes the expression of osteogenic genes. Concurrently, the predominant Ti(0002) crystal plane of FG Ti induces the formation of hydrophilic anatase titanium oxide layers, which accelerate biomineralization. In conclusion, this bioinspired FG Ti not only proves to show mechanical and bone-regenerative improvements but it also provides a new strategy for the future design of metallic biomaterials.

Tumor-associated macrophages induce inflammation and drug resistance in a mechanically tunable engineered model of osteosarcoma.

The tumor microenvironment is a complex and dynamic ecosystem composed of various physical cues and biochemical signals that facilitate cancer progression, and tumor-associated macrophages are especially of interest as a treatable target due to their diverse pro-tumorigenic functions. Engineered three-dimensional models of tumors more effectively mimic the tumor microenvironment than monolayer cultures and can serve as a platform for investigating specific aspects of tumor biology within a controlled setting. To study the combinatorial effects of tumor-associated macrophages and microenvironment mechanical properties on osteosarcoma, we co-cultured human osteosarcoma cells with macrophages within biomaterials-based bone tumor niches with tunable stiffness. In the first 24 h of direct interaction between the two cell types, macrophages induced an inflammatory environment consisting of high concentrations of tumor necrosis factor alpha (TNFα) and interleukin (IL)-6 within moderately stiff scaffolds. Expression of Yes-associated protein (YAP), but not its homolog, transcriptional activator with PDZ-binding motif (TAZ), in osteosarcoma cells was significantly higher than in macrophages, and co-culture of the two cells slightly upregulated YAP in both cells, although not to a significant degree. Resistance to doxorubicin treatment in osteosarcoma cells was correlated with inflammation in the microenvironment, and signal transducer and activator of transcription 3 (STAT3) inhibition diminished the inflammation-related differences in drug resistance but ultimately did not improve the efficacy of doxorubicin. This work highlights that the biochemical cues conferred by tumor-associated macrophages in osteosarcoma are highly variable, and signals derived from the immune system should be considered in the development and testing of novel drugs for cancer.

Nanocomposite Bioprinting for Tissue Engineering Applications.

Bioprinting aims to provide new avenues for regenerating damaged human tissues through the controlled printing of live cells and biocompatible materials that can function therapeutically. Polymeric hydrogels are commonly investigated ink materials for 3D and 4D bioprinting applications, as they can contain intrinsic properties relative to those of the native tissue extracellular matrix and can be printed to produce scaffolds of hierarchical organization. The incorporation of nanoscale material additives, such as nanoparticles, to the bulk of inks, has allowed for significant tunability of the mechanical, biological, structural, and physicochemical material properties during and after printing. The modulatory and biological effects of nanoparticles as bioink additives can derive from their shape, size, surface chemistry, concentration, and/or material source, making many configurations of nanoparticle additives of high interest to be thoroughly investigated for the improved design of bioactive tissue engineering constructs. This paper aims to review the incorporation of nanoparticles, as well as other nanoscale additive materials, to printable bioinks for tissue engineering applications, specifically bone, cartilage, dental, and cardiovascular tissues. An overview of the various bioinks and their classifications will be discussed with emphasis on cellular and mechanical material interactions, as well the various bioink formulation methodologies for 3D and 4D bioprinting techniques. The current advances and limitations within the field will be highlighted.