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Calcium phosphate cement (CPC) has evolved as an appealing bone substitute material, especially since CPCs were combined with poly(lactic-co-glycolic acid) (PLGA) porogens to render the resulting CPC/PLGA composite degradable. In view of the multiple variables of CPC and PLGA used previously, the effect of CPC composition and PLGA porogen morphology (i.e., microspheres versus microparticles) on the biological performance of CPC/PLGA has not yet been investigated. Consequently, we here aimed to evaluate comparatively various CPC/PLGA formulations varying in CPC composition and PLGA porogen morphology on their performance in a rabbit femoral condyle bone defect model. CPCs with a composition of 85 wt% α-TCP, 15 wt% dicalcium phosphate anhydrate (DCPA) and 5 wt% precipitated hydroxyapatite (pHA), or 100 wt% α-TCP were combined with spherical or irregularly shaped PLGA porogens (CPC/PLGA ratio of 60:40 wt% for all formulations). All CPC/PLGA formulations were applied via injection in bone defects, as created in the femoral condyle of rabbits, and retrieved for histological evaluation after 6 and 12 weeks of implantation. Descriptive histology and quantitative histomorphometry (i.e., material degradation and new bone formation) were used for analyses. Descriptively, all CPC/PLGA formulations showed material degradation at the periphery of the cement within 6 weeks of implantation. After 12 weeks, bone formation was observed extending into the defect core, replacing the degraded CPC/PLGA material. Quantitatively, similar material degradation (up to 87%) and new bone formation (up to 28%) values were observed, irrespective of compositional variations of CPC/PLGA formulations. These data prove that neither the CPC compositions nor the PLGA porogen morphologies as used in this work affect the biological performance of CPC/PLGA formulations in a rabbit femoral condyle bone defect model., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

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., (©2024 American Association for Cancer Research.)

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.

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Eleonora Dondossola reports financial support was provided by The Prostate Cancer SPORE (P50 CA140388–07). Eleonora Dondossola reports financial support and equipment, drugs, or supplies were provided by Bayer HealthCare Pharmaceuticals (57440)., (Copyright © 2024 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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., Competing Interests: No competing financial interests exist., (Copyright 2024, Mary Ann Liebert, Inc., publishers.)

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., (© The Author(s) 2024. Published by Oxford University Press.)

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 Tyr 270 . 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.

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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

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., (© 2023. The Author(s).)

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/cm 2 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., (© The Author(s) 2023. Published by Oxford University Press.)

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., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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., (Copyright © 2023. Published by Elsevier B.V.)

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.

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., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The aim of this preclinical study was to test the applicability of calcium phosphate cement (CPC)-poly(lactic-co-glycolic acid) (PLGA)-carboxymethylcellulose (CMC) as a bone substitute material for guided bone regeneration (GBR) procedures in a clinically relevant mandibular defect model in minipigs. In the study, a predicate device (i.e., BioOss ® ) was included for comparison. Critical-sized circular mandibular bone defects were created and filled with either CPC-PLGA-CMC without coverage with a GBR membrane or BioOss covered with a GBR membrane and left to heal for 4 and 12 weeks to obtain temporal insight in material degradation and bone formation. Bone formation increased significantly for both CPC-PLGA-CMC and BioOss with increasing implantation time. Further, no significant differences were found for bone formation at either 4 or 12 weeks between CPC-PLGA-CMC and BioOss. Finally, bone substitute material degradation increased significantly for both CPC-PLGA-CMC and BioOss from 4 to 12 weeks of implantation, showing the highest degradation for CPC-PLGA-CMC (∼85%) compared to BioOss (∼12%). In conclusion, this minipig study showed that CPC-PLGA-CMC can be used as a bone-grafting material and stimulates bone regeneration to a comparable extent as with BioOss particles. Importantly, CPC-PLGA-CMC degrades faster compared to BioOss, is easier to apply into a bone defect, and does not need the use of an additional GBR membrane. Consequently, the data support the further investigation of CPC-PLGA-CMC in human clinical trials. Impact statement Guided bone regeneration (GBR) is a frequently used dental surgical technique to regenerate the alveolar ridge to allow stable implant installation. However, stabilization of the GBR membrane and avoidance of bone graft movement remain a challenge. Consequently, there is need for the development of alternative materials to be used in GBR procedures that are easier to apply and induce predictable bone regeneration. In this minipig study, we focused on the applicability of calcium phosphate cement-poly(lactic-co-glycolic acid)-carboxymethylcellulose as an alternative bone substitute material for GBR procedures without the need of an additional GBR membrane.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Watson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Competing interests: The authors declare that they have no competing interests., (Copyright © 2023 Emma Watson and Antonios G. Mikos.)

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.

Extrusion bioprinted constructs for osteochondral tissue engineering were fabricated to study the effect of multi-material architecture on encapsulated human mesenchymal stem cells' tissue-specific matrix deposition and integration into an ex vivo porcine osteochondral explant model. Two extrusion fiber architecture groups with differing transition regions and degrees of bone- and cartilage-like bioink mixing were employed. The gradient fiber (G-Fib) architecture group showed an increase in chondral integration over time, 18.5 ± 0.7 kPa on Day 21 compared to 9.6 ± 1.6 kPa on Day 1 for the required peak push-out force, and the segmented fiber (S-Fib) architecture group did not, which corresponded to the increase in sulfated glycosaminoglycan deposition noted only in the G-Fib group and the staining for cellularity and tissue-specific matrix deposition at the fiber-defect boundary. Conversely, the S-Fib architecture was associated with significant mineralization over time, but the G-Fib architecture was not. Notably, both fiber groups also had similar chondral integration as a re-inserted osteochondral tissue control. While architecture did dictate differences in the cells' responses to their environment, architecture was not shown to distinguish a statistically significant difference in tissue integration via fiber push-out testing within a given time point or explant region. Use of this three-week osteochondral model demonstrates that these bioink formulations support the fabrication of cell-laden constructs that integrate into explanted tissue as capably as natural tissue and encapsulate osteochondral matrix-producing cells, and it also highlights the important role that spatial architecture plays in the engineering of multi-phasic tissue environments. STATEMENT OF SIGNIFICANCE: Here, an ex vivo model was used to interrogate fundamental questions about the effect of multi-material scaffold architectural choices on osteochondral tissue integration. Cell-encapsulating constructs resembling stratified osteochondral tissue were 3D printed with architecture consisting of either gradient transitions or segmented transitions between the bone-like and cartilage-like bioink regions. The printed constructs were assessed alongside re-inserted natural tissue plugs via mechanical tissue integration push-out testing, biochemical assays, and histology. Differences in osteochondral matrix deposition were observed based on architecture, and both printed groups demonstrated cartilage integration similar to the native tissue plug group. As 3D printing becomes commonplace within biomaterials and tissue engineering, this work illustrates critical 3D co-culture interactions and demonstrates the importance of considering architecture when interpreting the results of studies utilizing spatially complex, multi-material scaffolds., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies., (© 2022 Wiley Periodicals LLC.)