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There is a clinical need for osteochondral scaffolds with complex geometries for restoring articulating joint surfaces. To address that need, 3D-printing has enabled scaffolds to be created with anatomically shaped geometries and interconnected internal architectures, going beyond simple plug-shaped scaffolds that are limited to small, cylindrical, focal defects. A key challenge for restoring articulating joint surfaces with 3D-printed constructs is the mechanical loading environment, particularly to withstand delamination or mechanical failure. Although the mechanical performance of interfacial scaffolds is essential, interface strength testing has rarely been emphasized in prior studies with stratified scaffolds. In the pioneering studies where interface strength was assessed, varying methods were employed, which has made direct comparisons difficult. Therefore, the current review focused on 3D-printed scaffolds for osteochondral applications with an emphasis on interface integration and biomechanical evaluation. This 3D-printing focus included both multiphasic cylindrical scaffolds and anatomically shaped scaffolds. Combinations of different 3D-printing methods (e.g., fused deposition modeling, stereolithography, bioprinting with pneumatic extrusion of cell-laden hydrogels) have been employed in a handful of studies to integrate osteoinductive and chondroinductive regions into a single scaffold. Most 3D-printed multiphasic structures utilized either an interdigitating or a mechanical interlocking design to strengthen the construct interface and to prevent delamination during function. The most effective approach to combine phases may be to infill a robust 3D-printed osteal polymer with an interlocking chondral phase hydrogel. Mechanical interlocking is therefore recommended for scaling up multiphasic scaffold applications to larger anatomically shaped joint surface regeneration. For the evaluation of layer integration, the interface shear test is recommended to avoid artifacts or variability that may be associated with alternative approaches that require adhesives or mechanical grips. The 3D-printing literature with interfacial scaffolds provides a compelling foundation for continued work toward successful regeneration of injured or diseased osteochondral tissues in load-bearing joints such as the knee, hip, or temporomandibular joint., (© 2024 The Author(s). Journal of Biomedical Materials Research Part A published by Wiley Periodicals LLC.)

Translation of small-diameter tissue-engineered vascular grafts (TEVGs) for the treatment of coronary artery disease (CAD) remains an unfulfilled promise. This is largely due to the limited integration of TEVGs into the native vascular wall-a process hampered by the insufficient smooth muscle cell (SMC) infiltration and extracellular matrix deposition, and low vasoactivity. These processes can be promoted through the judicious modulation of the SMC toward a synthetic phenotype to promote remodeling and vascular integration; however, the expression of synthetic markers is often accompanied by a decrease in the expression of contractile proteins. Therefore, techniques that can precisely modulate the SMC phenotypical behavior could have the potential to advance the translation of TEVGs. In this review, we describe the phenotypic diversity of SMCs and the different environmental cues that allow the modulation of SMC gene expression. Furthermore, we describe the emerging biomaterial approaches to modulate the SMC phenotype in TEVG design and discuss the limitations of current techniques. In addition, we found that current studies in tissue engineering limit the analysis of the SMC phenotype to a few markers, which are often the characteristic of early differentiation only. This limited scope has reduced the potential of tissue engineering to modulate the SMC toward specific behaviors and applications. Therefore, we recommend using the techniques presented in this review, in addition to modern single-cell proteomics analysis techniques to comprehensively characterize the phenotypic modulation of SMCs. Expanding the holistic potential of SMC modulation presents a great opportunity to advance the translation of living conduits for CAD therapeutics.

Regenerative medicine approaches to restore the mandibular condyle of the temporomandibular joint (TMJ) may fill an unmet patient need. In this study, a method to implant an acellular regenerative TMJ prosthesis was developed for orthotopic implantation in a pilot goat study. The scaffold incorporated a porous, polycaprolactone-hydroxyapatite (PCL-HAp, 20wt% HAp) 3D printed condyle with a cartilage-matrix-containing hydrogel. A series of material characterizations was used to determine the structure, fluid transport, and mechanical properties of 3D printed PCL-HAp. To promote marrow uptake for cell seeding, a scaffold pore size of 152 ± 68 μm resulted in a whole blood transport initial velocity of 3.7 ± 1.2 mm·s -1 transported to the full 1 cm height. The Young's modulus of PCL was increased by 67% with the addition of HAp, resulting in a stiffness of 269 ± 20 MPa for etched PCL-HAp. In addition, the bending modulus increased by 2.06-fold with the addition of HAp to 470 MPa for PCL-HAp. The prosthesis design with an integrated hydrogel was compared with unoperated contralateral control and no-hydrogel group in a goat model for 6 months. A guide was used to make the condylectomy cut, and the TMJ disc was preserved. MicroCT assessment of bone suggested variable tissue responses with some regions of bone growth and loss, although more loss may have been exhibited by the hydrogel group than the no-hydrogel group. A benchtop load transmission test suggested that the prosthesis was not shielding load to the underlying bone. Although variable, signs of neocartilage formation were exhibited by Alcian blue and collagen II staining on the anterior, functional surface of the condyle. Overall, this study demonstrated signs of functional TMJ restoration with an acellular prosthesis. There were apparent limitations to continuous, reproducible bone formation, and stratified zonal cartilage regeneration. Future work may refine the prosthesis design for a regenerative TMJ prosthesis amenable to clinical translation.

Hydrogel mechanical properties for tissue engineering are often reported in terms of a compressive elastic modulus derived from a linear regression of a typically non-linear stress-strain plot. There is a need for an alternative model to fit the full strain range of tissue engineering hydrogels. Fortunately, the Ogden model provides a shear modulus, μ 0 , and a nonlinear parameter, α, for routine analysis of compression to failure. Three example hydrogels were tested: (1) pentenoate-modified hyaluronic acid (PHA), (2) dual-crosslinked PHA and polyethylene glycol diacrylate (PHA-PEGDA), and (3) composite PHA-PEGDA hydrogel with cryoground devitalized cartilage (DVC) at 5, 10, and 15%w/v concentration (DVC5, DVC10, and DVC15, respectively). Gene expression analyses suggested that the DVC hydrogels supported chondrogenesis of human bone marrow mesenchymal stem cells to some degree. Both linear regression (5 to 15% strain) and Ogden fits (to failure) were performed. The compressive elastic modulus, E, was over 4-fold higher in the DVC15 group relative to the PHA group (129 kPa). Similarly, the shear modulus, μ 0 , was over 3-fold higher in the DVC15 group relative to the PHA group (37 kPa). The PHA group exhibited a much higher degree of nonlinearity (α = 10) compared to the DVC15 group (α = 1.4). DVC hydrogels may provide baseline targets of μ 0 and α for future cartilage tissue engineering studies. The Ogden model was demonstrated to fit the full strain range with high accuracy (R 2  = 0.998 ± 0.001) and to quantify nonlinearity. The current study provides an Ogden model as an attractive alternative to the elastic modulus for tissue engineering constructs., 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.)

Regeneration of calvarial bone remains a major challenge in the clinic as available options do not sufficiently regenerate bone in larger defect sizes. Calvarial bone regeneration cases involving secondary medical conditions, such as brain herniation during traumatic brain injury (TBI) treatment, further exacerbate treatment options. Hydrogels are well-positioned for severe TBI treatment, given their innate flexibility and potential for bone regeneration to treat TBI in a single-stage surgery. The current study evaluated a photocrosslinking pentenoate-modified hyaluronic acid polymer with thiolated demineralized bone matrix (i.e., TDBM hydrogel) capable of forming a completely interconnected hydrogel matrix for calvarial bone regeneration. The TDBM hydrogel demonstrated a setting time of 120 s, working time of 3 to 7 days, negligible change in setting temperature, physiological setting pH, and negligible cytotoxicity, illustrating suitable performance for in vivo application. Side-by-side ovine calvarial bone defects (19 mm diameter) were employed to compare the TDBM hydrogel to the standard-of-care control material DBX ® . After 16 weeks, the TDBM hydrogel had comparable healing to DBX ® as demonstrated by mechanical push-out testing (~800 N) and histology. Although DBX ® had 59% greater new bone volume compared to the TDBM hydrogel via micro-computed tomography, both demonstrated minimal bone regeneration overall (15 to 25% of defect volume). The current work presents a method for comparing the regenerative potential of new materials to clinical products using a side-by-side cranial bone defect model. Comparison of novel biomaterials to a clinical product control (i.e., standard-of-care) provides an important baseline for successful regeneration and potential for clinical translation., Competing Interests: Declaration of interests Dr. Jakob Townsend and Dr. Michael Detamore are co-owners of Leefa Biotech, LLC.

Scaffolds derived from cartilage extracellular matrix may contain intrinsic chondroinductivity and have promise for cartilage regeneration. Cartilage is typically ground into devitalized particles (DVC) and several groups have pioneered innovative methods to rebuild the DVC into a new scaffold. However, challenges remain regarding the fluid and solid biomechanics of cartilage-based scaffolds in achieving 1) high mechanical performance akin to native cartilage and 2) easy surgical delivery/retention. Fortunately, photocrosslinking bioinks may benefit clinical translation: paste-like/injectable precursor rheology facilitates surgical placement, and in situ photocrosslinking enables material retention within any size/shape of defect. While solubilized DVC has been modified with methacryloyls (MeSDVC), MeSDVC is limited by slow crosslinking times (e.g., 5-10 min). Therefore, in the current study, we fabricated a pentenoate-modified SDVC (PSDVC), to enable a faster crosslinking reaction via a thiol-ene click chemistry. The crosslinking time of the PSDVC was faster (∼1.7 min) than MeSDVC (∼4 min). We characterized the solid and fluid mechanics/printabilities of PSDVC, pentenoate-modified hyaluronic acid (PHA), and the PHA or PSDVC with added DVC particles. While the addition of DVC particles enhanced the printed shape fidelity of PHA or PSDVC, the increased clogging decreased the ease of printing and cell viability after bioprinting, and future refinement is needed for DVC-containing bioinks. However, the PSDVC alone had a paste-like rheology/good bioprintability prior to crosslinking, the fastest crosslinking time (i.e., 1.7 min), and the highest compressive modulus (i.e., 3.12 ± 0.41 MPa) after crosslinking. Overall, the PSDVC may have future potential as a translational material for cartilage repair., 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.)

Hyaluronic acid (HA) hydrogels have been used for a multitude of applications, perhaps most notably for tissue engineering and regenerative medicine, owing to the versatility of the polymer and its tunable nature. Various groups have investigated the impact of hydrogel parameters (e.g. molecular weight, concentration, stiffness, etc) in vitro and in vivo to achieve desired material performance characteristics. A limitation in the literature to date has been that altering one hydrogel parameter (a 'manipulated variable') to achieve a given hydrogel characteristic (a 'controlled variable') changes two variables at a time (e.g. altering molecular weight and/or concentration to investigate cell response to stiffness). Therefore, if cell responses differ, it may be possible that more than one variable caused the changes in observed responses. In the current study, we leveraged thiol-ene click chemistry with a crosslinker to develop a method that minimizes material performance changes and permitted multiple material properties to be independently held constant to evaluate a single variable at a time. Independent control was accomplished by tuning the concentration of crosslinker to achieve an effectively constant stiffness for different HA hydrogel molecular weights and polymer concentrations. Specific formulations were thereby identified that enabled the molecular weight (76-1550 kDa), concentration (2%-10%), or stiffness (∼1-350 kPa) to be varied while the other two were held constant, a key technical achievement. The response of rat mesenchymal stem cells to varying molecular weight, concentration, and stiffness demonstrated consistent upregulation of osteocalcin gene expression. The methodology presented to achieve independent control of hydrogel parameters may potentially be adopted by others for alternative hydrogel polymers, cell types, or cell culture medium compositions to minimize confounding variables in experimental hydrogel designs., (© 2022 IOP Publishing Ltd.)

Inducing and maintaining a hyaline cartilage phenotype are the greatest challenge for cartilage regeneration. Synthetic chondroinductive biomaterials might be the answer to the unmet clinical need for a safe, stable, and cost-effective material capable of inducing true hyaline cartilage formation. The past decade witnessed an emergence of peptides to achieve chondrogenesis, as peptides have the advantages of versatility, high target specificity, minimized toxicity and immunogenicity, and ease of synthesis. In this study, we review peptides as the basis for creating promising synthetic chondroinductive biomaterials for in situ scaffold-based cartilage regeneration. We provide a thorough review of peptides evaluated for cartilage regeneration while distinguishing between peptides reported to induce chondrogenesis independently, and peptides reported to act in synergy with other growth factors to induce cartilage regeneration. In addition, we highlight that most peptide studies have been in vitro , and appropriate controls are not always present. A few rigorously performed in vitro studies have proceeded to in vivo studies, but the peptides in those in vivo studies were mainly introduced through systemic, subcutaneous, or intra-articular injections, with a paucity of studies employing in situ defects with appropriate controls. Clinical translation of peptides will require the evaluation of these peptides in well-controlled in vivo cartilage defect studies. In the decade ahead, we may be poised to leverage peptides to design devices that are safe, reproducible, cost-efficient, and scalable biomaterials, which are themselves chondroinductive to achieve true hyaline cartilage regeneration without the need for growth factors and other small molecules. Impact statement The regeneration of articular cartilage into its original structural, functional, and organizational hyaline phenotype remains a significant problem in the tissue engineering and orthopedic community. While cell-based solutions have shown promising outcomes, there are realistic translational challenges inherent to cell therapies. Alternatively, biomaterials have been widely studied and used as scaffolds to support and facilitate cartilage regeneration; however, the key technical challenge is to independently induce cartilage regeneration. The search for chondroinductive compounds and materials is an emerging area of research with peptides at its heart, which presents a timely opportunity to review and highlight peptides with cartilage regenerative activity and to fill gaps from previous reviews. The content of this review will serve as a valuable guide for researchers pursuing the discovery of new chondroinductive peptides or looking into incorporating the most promising existing peptides in their work.

The potential chondroinductivity from cartilage matrix makes it promising for cartilage repair; however, cartilage matrix-based hydrogels developed thus far have failed to match the mechanical performance of native cartilage or be bioprinted without adding polymers for reinforcement. There is a need for cartilage matrix-based hydrogels with robust mechanical performance and paste-like precursor rheology for bioprinting/enhanced surgical placement. In the current study, our goals were to increase hydrogel stiffness and develop the paste-like precursor/printability of our methacryl-modified solubilized and devitalized cartilage (MeSDVC) hydrogels. We compared two methacryloylating reagents, methacrylic anhydride (MA) and glycidyl methacrylate (GM), and varied the molar excess (ME) of MA from 2 to 20. The MA-modified MeSDVCs had greater methacryloylation than GM-modified MeSDVC (20 ME). While GM and most of the MA hydrogel precursors exhibited paste-like rheology, the 2 ME MA and GM MeSDVCs had the best printability (i.e., shape fidelity, filament collapse). After crosslinking, the 2 ME MA MeSDVC had the highest stiffness (1.55 ± 0.23 MPa), approaching the modulus of native cartilage, and supported the viability/adhesion of seeded cells for 15 days. Overall, the MA (2 ME) improved methacryloylation, hydrogel stiffness, and printability, resulting in a stand-alone MeSDVC printable biomaterial. The MeSDVC has potential as a future bioink and has future clinical relevance for cartilage repair.

Manual tissue decellularization is an onerous process that requires the application of many sequential treatments by an operator and can be prone to user error and result variability. While automated decellularization devices have been previously reported, with advances being made in recent years toward open-source platforms, previous automated decellularization devices have been reliant on hardware or software components that are closed-source and proprietary. The aim of the current work was to develop and validate a full open-source automated decellularization system to be available for others to adopt. The open-source decellularization apparatus is a low-cost (<$2000) device that may easily be adapted to an array of decellularization protocols, with an example parts' list provided herein. The automated decellularization device was used to decellularize hyaline cartilage, knee meniscus, and tendon tissues. Cartilage, meniscus, and tendon tissue demonstrated 97%, 99%, and 96% reduction in DNA content after decellularization, respectively, and with effective decellularization confirmed visually via histology. High retentions of glycosaminoglycans (GAGs), collagen, and other proteins were observed in meniscus and tendon following decellularization. Results with manual decellularization with meniscus tissue were consistent with the automated decellularization process. Decellularized cartilage (DCC) demonstrated a 34% decrease in GAG content, while the protein and collagen content did not significantly change. The current study demonstrated that native-like decellularized tissues were produced reproducibly using the reported open-source automated decellularization platform, providing an adoptable platform for production of decellularized tissues by others. Impact statement Decellularized extracellular matrix (ECM)-based materials are appealing for tissue engineering, but production of these materials is historically time-intensive, tedious, and prone to user error. Adoption of an automated system may be a barrier for many research groups due to cost and complexity. In this article, a low-cost open-source platform for automated decellularization is presented. This method is validated by decellularizing porcine musculoskeletal tissues and demonstrating the native-like compositional properties of these decellularized tissues. The ability to produce decellularized tissue in an automated manner is useful for further research of ECM-based materials and potential clinical applications.

There is growing evidence indicating the need to combine the rehabilitation and regenerative medicine fields to maximize functional recovery after spinal cord injury (SCI), but there are limited methods to synergistically combine the fields. Conductive biomaterials may enable synergistic combination of biomaterials with electric stimulation (ES), which may enable direct ES of neurons to enhance axon regeneration and reorganization for better functional recovery; however, there are three major challenges in developing conductive biomaterials: (1) low conductivity of conductive composites, (2) many conductive components are cytotoxic, and (3) many conductive biomaterials are pre-formed scaffolds and are not injectable. Pre-formed, noninjectable scaffolds may hinder clinical translation in a surgical context for the most common contusion-type of SCI. Alternatively, an injectable biomaterial, inspired by lessons from bioinks in the bioprinting field, may be more translational for contusion SCIs. Therefore, in the current study, a conductive hydrogel was developed by incorporating high aspect ratio citrate-gold nanorods (GNRs) into a hyaluronic acid and gelatin hydrogel. To fabricate nontoxic citrate-GNRs, a robust synthesis for high aspect ratio GNRs was combined with an indirect ligand exchange to exchange a cytotoxic surfactant for nontoxic citrate. For enhanced surgical placement, the hydrogel precursor solution (i.e., before crosslinking) was paste-like, injectable/bioprintable, and fast-crosslinking (i.e., 4 min). Finally, the crosslinked hydrogel supported the adhesion/viability of seeded rat neural stem cells in vitro. The current study developed and characterized a GNR conductive hydrogel/bioink that provided a refinable and translational platform for future synergistic combination with ES to improve functional recovery after SCI., (© 2021 Wiley Periodicals LLC.)

The individual approaches of regenerative medicine efforts alone and rehabilitation efforts alone have not yet fully restored function after severe spinal cord injury (SCI). Regenerative rehabilitation may be leveraged to promote regeneration of the spinal cord tissue, and promote reorganization of the regenerated neural pathways and intact spinal circuits for better functional recovery for SCI. Conductive biomaterials may be a linchpin that empowers the synergy between regenerative medicine and rehabilitation approaches, as electrical stimulation applied to the spinal cord could facilitate neural reorganization. In this review, we discuss current regenerative medicine approaches in clinical trials and the rehabilitation, or neuromodulation, approaches for SCI, along with their respective translational limitations. Furthermore, we review the translational potential, in a surgical context, of conductive biomaterials (e.g., conductive polymers, carbon-based materials, metallic nanoparticle-based materials) as they pertain to SCI. While pre-formed scaffolds may be difficult to translate to human contusion SCIs, injectable composites that contain blended conductive components and can form within the injury may be more translational. However, given that there are currently no in vivo SCI studies that evaluated conductive materials combined with rehabilitation approaches, we discuss several limitations of conductive biomaterials, including demonstrating safety and efficacy, that will need to be addressed in the future for conductive biomaterials to become SCI therapeutics. Even so, the use of conductive biomaterials creates a synergistic opportunity to merge the fields of regenerative medicine and rehabilitation and redefine what regenerative rehabilitation means for the spinal cord. STATEMENT OF SIGNIFICANCE: For spinal cord injury (SCI), the individual approaches of regenerative medicine and rehabilitation are insufficient to fully restore functional recovery; however, the goal of regenerative rehabilitation is to combine these two disparate fields to maximize the functional outcomes. Concepts similar to regenerative rehabilitation for SCI have been discussed in several reviews, but for the first time, this review considers how conductive biomaterials may synergize the two approaches. We cover current regenerative medicine and rehabilitation approaches for SCI, and the translational advantages and disadvantages, in a surgical context, of conductive biomaterials used in biomedical applications that may be additionally applied to SCI. Furthermore, we identify the current limitations and translational challenges for conductive biomaterials before they may become therapeutics for SCI., 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 © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Tracheal tissue engineering has become an active area of interest among clinical and scientific communities; however, methods to evaluate success of in vivo tissue-engineered solutions remain primarily qualitative. These evaluation methods have generally relied on the use of photographs to qualitatively demonstrate tracheal patency, endoscopy to image healing over time, and histology to determine the quality of the regenerated extracellular matrix. Although those generally qualitative methods are valuable, they alone may be insufficient. Therefore, to quantitatively assess tracheal regeneration, we recommend the inclusion of microcomputed tomography (μCT) to quantify tracheal patency as a standard outcome analysis. To establish a standard of practice for quantitative μCT assessment for tracheal tissue engineering, we recommend selecting a constant length to quantify airway volume. Dividing airway volumes by a constant length provides an average cross-sectional area for comparing groups. We caution against selecting a length that is unjustifiably large, which may result in artificially inflating the average cross-sectional area and thereby diminishing the ability to detect actual differences between a test group and a healthy control. Therefore, we recommend selecting a length for μCT assessment that corresponds to the length of the defect region. We further recommend quantifying the minimum cross-sectional area, which does not depend on the length, but has functional implications for breathing. We present empirical data to elucidate the rationale for these recommendations. These empirical data may at first glance appear as expected and unsurprising. However, these standard methods for performing μCT and presentation of results do not yet exist in the literature, and are necessary to improve reporting within the field. Quantitative analyses will better enable comparisons between future publications within the tracheal tissue engineering community and empower a more rigorous assessment of results. Impact statement The current study argues for the standardization of microcomputed tomography (μCT) as a quantitative method for evaluating tracheal tissue-engineered solutions in vivo or ex vivo. The field of tracheal tissue engineering has generally relied on the use of qualitative methods for determining tracheal patency. A standardized quantitative evaluation method currently does not exist. The standardization of μCT for evaluation of in vivo studies would enable a more robust characterization and allow comparisons between groups within the field. The impact of standardized methods within the tracheal tissue engineering field as presented in the current study would greatly improve the quality of published work.

Biological interactions, toxicity, and environmental fate of engineered nanoparticles are affected by colloidal stability and aggregation. To assess nanoparticle aggregation, analytical methods are needed that allow quantification of individual nanoparticle aggregates. However, most techniques used for nanoparticle aggregation analysis are limited to ensemble measurements or require harsh sample preparation that may introduce artifacts. An ideal method would analyze aggregate size in situ with single-nanoparticle resolution. Here, we established and validated single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS) as an unbiased high-throughput analytical technique to quantify nanoparticle size distributions and aggregation in situ. We induced nanoparticle aggregation by exposure to physiologically relevant saline conditions and applied SP-ICP-MS to quantify aggregate size and aggregation kinetics at the individual aggregate level. In situ SP-ICP-MS analysis revealed rational surface engineering principles for the preparation of colloidally stable nanoparticles. Our quantitative SP-ICP-MS technique is a platform technology to evaluate aggregation characteristics of various types of surface-engineered nanoparticles under physiologically relevant conditions. Potential widespread applications of this method may include the study of nanoparticle aggregation in environmental samples and the preparation of colloidally stable nanoparticle formulations for bioanalytical assays and nanomedicine. Graphical abstract.

Bone regeneration of large cranial defects, potentially including traumatic brain injury (TBI) treatment, presents a major problem with non-crosslinking, clinically available products due to material migration outside the defect. Commercial products such as bone cements are permanent and thus not conducive to bone regeneration, and typical commercial bioactive materials for bone regeneration do not crosslink. Our previous work demonstrated that non-crosslinking materials may be prone to material migration following surgical placement, and the current study attempted to address these problems by introducing a new hydrogel system where tissue particles are themselves the crosslinker. Specifically, a pentenoate-modified hyaluronic acid (PHA) polymer was covalently linked to thiolated tissue particles of demineralized bone matrix (TDBM) or devitalized tendon (TDVT), thereby forming an interconnected hydrogel matrix for calvarial bone regeneration. All hydrogel precursor solutions exhibited sufficient yield stress for surgical placement and an adequate compressive modulus post-crosslinking. Critical-size calvarial defects were filled with a 4% PHA hydrogel containing 10 or 20% TDBM or TDVT, with the clinical product DBX Ⓡ being employed as the standard of care control for the in vivo study. At 12 weeks, micro-computed tomography analysis demonstrated similar bone regeneration among the experimental groups, TDBM and TDVT, and the standard of care control DBX Ⓡ . The group with 10% TDBM was therefore identified as an attractive material for potential calvarial defect repair, as it additionally exhibited a sufficient initial recovery after shearing (i.e., > 80% recovery). Future studies will focus on applying a hydrogel in a rat model for treatment of TBI. STATEMENT OF SIGNIFICANCE: Non-crosslinking materials may be prone to material migration from a calvarial bone defect following surgical placement, which is problematic for materials intended for bone regeneration. Unfortunately, typical crosslinking materials such as bone cements are permanent and thus not conducive to bone regeneration, and typical bioactive materials for bone regeneration such as tissue matrix are not crosslinked in commercial products. The current study addressed these problems by introducing a new biomaterial where tissue particles are themselves the crosslinker in a hydrogel system. The current study successfully demonstrated a new material based on pentenoate-modified hyaluronic acid with thiolated demineralized bone matrix that is capable of rapid crosslinking, with desirable paste-like rheology of the precursor material for surgical placement, and with bone regeneration comparable to a commercially available standard-of-care product. Such a material may hold promise for a single-surgery treatment of severe traumatic brain injury (TBI) following hemicraniectomy., 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 © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Difficulty breathing due to tracheal stenosis (i.e. narrowed airway) diminishes the quality of life and can potentially be life-threatening. Tracheal stenosis can be caused by congenital anomalies, external trauma, infection, intubation-related injury, and tumors. Common treatment methods for tracheal stenosis requiring surgical intervention include end-to-end anastomosis, slide tracheoplasty and/or laryngotracheal reconstruction. Although the current methods have demonstrated promise for treatment of tracheal stenosis, a clear need exists for the development of new biomaterials that can hold the trachea open after the stenosed region has been surgically opened, and that can support healing without the need to harvest autologous tissue from the patient. The current study therefore evaluated the use of electrospun nanofiber scaffolds encapsulating 3D-printed PCL rings to patch induced defects in rabbit tracheas. The nanofibers were a blend of polycaprolactone (PCL) and polylactide-co-caprolactone (PLCL), and encapsulated either the cell adhesion peptide, RGD, or antimicrobial compound, ceragenin-131 (CSA). Blank PCL/PLCL and PCL were employed as control groups. Electrospun patches were evaluated in a rabbit tracheal defect model for 12 weeks, which demonstrated re-epithelialization of the luminal side of the defect. No significant difference in lumen volume was observed for the PCL/PLCL patches compared to the uninjured positive control. Only the RGD group did not lead to a significant decrease in the minimum cross-sectional area compared to the uninjured positive control. CSA reduced bacteria growth in vitro, but did not add clear value in vivo. Adequate tissue in-growth into the patches and minimal tissue overgrowth was observed inside the patch material. Areas of future investigation include tuning the material degradation time to balance cell adhesion and structural integrity.

Three-dimensional (3D) bioprinting holds the promise to fabricate tissue and organ substitutes for regenerative medicine. However, the lack of bioactive inks to fabricate and support functional living constructs is one of the main limitations hindering the progress of this technology. In this study, a biofunctional human-based nanocomposite bioink (HUink) composed of platelet lysate hydrogels reinforced by cellulose nanocrystals is reported. When combined with suspended bioprinting technologies, HUink allows the biofabrication of 3D freeform constructs with high resolution and integrity, mimicking the hierarchical nano-to-macro fibrillary composition of native tissues. Remarkably, HUink supports bioprinting of stem cells with high viability immediately after extrusion and over long-term cell culture without the need for additional biochemical or animal-derived media supplementation. As opposed to typical polymer-based bioinks, the pool of growth factors, cytokines and adhesion proteins in HUink boosts cell spreading and proliferation, stimulating the fast production of cell-secreted extracellular matrix. This innovative bioprinting platform with unpaired biofunctionality allows the fabrication of complex freeform cell-laden constructs that can ultimately be applied in the development of xeno-free 3D tissue models for in vitro research or to develop tissue and organ surrogates for clinical applications.

A synthetic 'chondroinductive' biomaterial that could induce chondrogenesis without the need for growth factors, extracellular matrix, or pre-seeded cells could revolutionize orthopedic regenerative medicine. The objective of the current study was thus to introduce a synthetic SPPEPS peptide and evaluate its ability to induce chondrogenic differentiation. In the current study, dissolving a synthetic chondroinductive peptide candidate (100 ng/mL SPPEPS) in the culture medium of rat bone marrow-derived mesenchymal stem cells (rBMSCs) elevated collagen type II gene expression compared to the negative control (no growth factor or peptide in the cell culture medium) after 3 days. In addition, proteomic analyses indicated similarities in pathways and protein profiles between the positive control (10 ng/mL TGF-β 3 ) and peptide group (100 ng/mL SPPEPS), affirming the potential of the peptide for chondroinductivity. Incorporating the SPPEPS peptide in combination with the RGD peptide in pentenoate-functionalized hyaluronic acid (PHA) hydrogels elevated the collagen type II gene expression of the rBMSCs cultured on top of the hydrogels compared to using either peptide alone. The evidence suggests that SPPEPS may be a chondroinductive peptide, which may be enhanced in combination with an adhesion peptide.

Bioprinting technologies have tremendous potential for advancing regenerative medicine due to the precise spatial control over depositing a printable biomaterial, or bioink. Despite the growing interest in bioprinting, the field is challenged with developing biomaterials for extrusion-based bioprinting. The paradigm of contemporary bioink studies relies on trial-and-error methods for discovering printable biomaterials, which has little practical use for others who endeavor to develop bioinks. There is pressing need to follow the precedent set by a few pioneering studies that have attempted to standardize bioink characterizations for determining the properties that define printability. Here, we developed a pentenoate-functionalized hyaluronic acid hydrogel (PHA) into a printable bioink and used three recommended, quantitative rheological assessments to characterize the printability: 1) yield stress, 2) viscosity, and 3) storage modulus recovery. The most important characteristic is the yield stress; we found a yield stress upper limit of ∼1000 Pa for PHA. Measuring the viscosity was advantageous for determining shear-thinning behavior, which aided in extruding highly viscous PHA through a nozzle. Post-printing recovery is required to maintain shape fidelity and we found storage modulus recoveries above ∼85% were sufficient for PHA. Two formulations had superior printability (i.e., 1.5 MDa PHA - 4 wt%, and 1 MDa PHA - 8 wt%), and increasing cell concentrations in PHA up to 9 × 10 6  cells/mL had minimal effects on the printability. Even so, other factors such as sterilization and peptide modifications to enhance bioactivity may influence printability, highlighting the need for investigators to consider such factors when developing new bioinks. STATEMENT OF SIGNIFICANCE: Bioprinting has potential for regenerating damaged tissues; however, there are a limited number of printable biomaterials, and developing new bioinks is challenging because the required material physical properties for extrusion-based printing are not yet known. Most new bioinks are developed by trial-and-error, which is neither efficient nor comparable across materials. There is a need for the field to begin utilizing standard methods proposed by a few pioneering studies to characterize new bioinks. Therefore, we have developed the printability of a hyaluronic acid based-hydrogel and characterized the material with three quantitative rheological tests. The current work impacts the bioprinting field by demonstrating and encouraging the use of universal bioink characterizations and by providing printability windows to advance new bioink development., (Copyright © 2019 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

In the field of regenerative medicine, creating a biomaterial device with the potential alone to affect cellular fate is a desirable translational strategy. Native tissues and growth factors are attractive candidates to provide desired signals in a biomaterial environment. However, these molecules can have translational challenges such as high cost, complicated regulatory pathways, and/or limitations with reproducibility. In regenerative medicine, there is a burgeoning community of investigators who seek to overcome these challenges by introducing synthetic peptides to mimic the desirable signals provided by growth factors and tissue matrices. Since in cartilage tissue, cell-adhesion signaling mediates cell migration, growth, and differentiation, synthetic peptides that mimic a desired cell-adhesion sequence may help to control cellular fate. This review emphasizes the value of the signaling ability of peptides, specifically in the cartilage regeneration field. The primary challenge in cartilage regeneration is to regenerate true hyaline cartilage instead of a fibrous tissue. The vision is to create materials that take advantage of the signaling abilities of peptides and that themselves induce chondrogenesis without the need for tissue-derived matrix or growth factors, which could potentially revolutionize arthritis prevention and treatment.

Hydrogels - water swollen cross-linked networks - have demonstrated considerable promise in tissue engineering and regenerative medicine applications. However, ambiguity over which rheological properties are needed to characterize these gels before crosslinking still exists. Most hydrogel research focuses on the performance of the hydrogel construct after implantation, but for clinical practice, and for related applications such as bioinks for 3D bioprinting, the behavior of the pre-gelled state is also critical. Therefore, the goal of this review is to emphasize the need for better rheological characterization of hydrogel precursor formulations, and standardized testing for surgical placement or 3D bioprinting. In particular, we consider engineering paste or putty precursor solutions (i.e., suspensions with a yield stress), and distinguish between these differences to ease the path to clinical translation. The connection between rheology and surgical application as well as how the use of paste and putty nomenclature can help to qualitatively identify material properties are explained. Quantitative rheological properties for defining materials as either pastes or putties are proposed to enable easier adoption to current methods. Specifically, the three-parameter Herschel-Bulkley model is proposed as a suitable model to correlate experimental data and provide a basis for meaningful comparison between different materials. This model combines a yield stress, the critical parameter distinguishing solutions from pastes (100-2000 Pa) and from putties (>2000 Pa), with power law fluid behavior once the yield stress is exceeded. Overall, successful implementation of paste or putty handling properties to the hydrogel precursor may minimize the surgeon-technology learning time and ultimately ease incorporation into current practice. Furthermore, improved understanding and reporting of rheological properties will lead to better theoretical explanations of how materials affect rheological performances, to better predict and design the next generation of biomaterials.

Tracheal stenosis caused by congenital anomalies, tumors, trauma, or intubation-related damage can cause severe breathing issues, diminishing the quality of life, and potentially becoming fatal. Current treatment methods include laryngotracheal reconstruction or slide tracheoplasty. Laryngotracheal reconstruction utilizes rib cartilage harvested from the patient, requiring a second surgical site. Slide tracheoplasty involves a complex surgical procedure to splay open the trachea and reconnect both segments to widen the lumen. A clear need exists for new and innovative approaches that can be easily adopted by surgeons, and to avoid harvesting autologous tissue from the patient. This study evaluated the use of an electrospun patch, consisting of randomly layered polycaprolactone (PCL) nanofibers enveloping 3D-printed PCL rings, to create a mechanically robust, suturable, air-tight, and bioresorbable graft for the treatment of tracheal defects. The study design incorporated two distinct uses of PCL: electrospun fibers to promote tissue integration, while remaining air-tight when wet, and 3D-printed rings to hold the airway open and provide external support and protection during the healing process. Electrospun, reinforced tracheal patches were evaluated in an ovine model, in which all sheep survived for 10 weeks, although an overgrowth of fibrous tissue surrounding the patch was observed to significantly narrow the airway. Minimal tissue integration of the surrounding tissue and the electrospun fibers suggested the need for further improvement. Potential areas for further improvement include a faster degradation rate, agents to increase cellular adhesion, and/or an antibacterial coating to reduce the initial bacterial load.

Hydrogel surface properties can be modified to form bioactive interfaces to modulate the osteogenic differentiation of stem cells. In this work, a hydrogel made of gelatin methacrylamide (GelMA) and alginate was designed and tested as a scaffold to control stem-cell osteogenic differentiation. The hydrogel's surface was treated with polydopamine (pDA) to create an adhesive layer for the adsorption of the osteoinductive drug dexamethasone (Dex). The presence of the pDA coating enhanced Dex adsorption and retention over 21 days. This effect resulted in a delay in the osteogenic differentiation of hASCs cultured on the hydrogel treated with a pDA layer.

Traumatic brain injury (TBI) is a life-threatening condition defined by internal brain herniation. Severe TBI is commonly treated by a two-stage surgical intervention, where decompressive craniectomy is first conducted to remove a large portion of calvarial bone and allow unimpeded brain swelling. In the second surgery, spaced weeks to months after the first, cranioplasty is performed to restore the cranial bone. Hydrogels with paste-like precursor solutions for surgical placement may potentially revolutionize TBI treatment by permitting a single-stage surgical intervention, capable of being implanted with the initial surgery, remaining pliable during brain swelling, and tuned to regenerate calvarial bone after brain swelling has subsided. The current study evaluated the use of photocrosslinkable pentenoate-functionalized hyaluronic acid (PHA) and non-crosslinking hyaluronic acid (HA) hydrogels encapsulating naturally derived tissue particles of demineralized bone matrix (DBM), devitalized cartilage (DVC), devitalized meniscus (DVM), or devitalized tendon (DVT) for bone regeneration in critical-size rat calvarial defects. All hydrogel precursors exhibited a yield stress for placement and addition of particles increased the average material compressive modulus. The HA-DBM (4-30%), PHA (4%), and PHA-DVT (4-30%) groups had 5 (p < 0.0001), 3.1, and 3.2 (p < 0.05) times greater regenerated bone volume compared to the sham (untreated defect) group, respectively. In vitro cell studies suggested that the PHA-DVT (4-10%) group would have the most desirable performance. Overall, hydrogels containing DVT particles outperformed other materials in terms of bone regeneration in vivo and calcium deposition in vitro. Hydrogels containing DVT will be further evaluated in future rat TBI studies., Statement of Significance: Traumatic brain injury (TBI) is a life-threatening condition characterized by severe brain swelling and is currently treated by a two-stage surgical procedure. Complications associated with the two-stage surgical intervention include the occurrence of the condition termed syndrome of the trephined; however, the condition is completely reversible once the secondary surgery is performed. A desirable TBI treatment would include a single surgical intervention to avoid syndrome of the trephined altogether. The first hurdle in reaching the overall goal is to develop a pliable hydrogel material that can regenerate the patient's bone. The development of a pliable hydrogel technology would greatly impact the field of bone regeneration for TBI application and other areas of bone regeneration., (Copyright © 2018 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Introduction: Spheroids of mesenchymal stem cells (MSCs) in cartilage tissue engineering have been shown to enhance regenerative potential owing to their 3D structure. In this study, we explored the possibility of priming spheroids under different media to replace the use of inductive surface coatings for chondrogenic differentiation., Methods: Rat bone marrow-derived MSCs were organized into cell spheroids by the hanging drop technique and subsequently cultured on hyaluronic acid (HA) coated or non-coated well plates under different cell media conditions. Endpoint analysis included cell viability, DNA and Glycosaminoglycan (GAG) and collagen content, gene expression and immunohistochemistry., Results: For chondrogenic applications, MSC spheroids derived on non-coated surfaces outperformed the spheroids derived from HA-coated surfaces in matrix synthesis and collagen II gene expression. Spheroids on non-coated surfaces gave rise to the highest collagen and GAG when primed with medium containing insulin-like growth factor (IGF) for 1 week during spheroid formation. Spheroids that were grown in chondroinductive raw material-inclusive media such as aggrecan or chondroitin sulfate exhibited the highest Collagen II gene expression in the non-coated surface at 1 week., Conclusion: Media priming by growth factors and raw materials might be a more predictive influencer of chondrogenesis compared to inductive-surfaces. Such tailored bioactivity of the stem cell spheroids in the stage of the spheroid formation may give rise to a platform technology that may eventually produce spheroids capable of chondrogenesis achieved by mere media manipulation, skipping the need for additional culture on a modified surface, that paves the way for cost-effective technologies.

In the treatment of severe traumatic brain injury (TBI), decompressive craniectomy is commonly used to remove a large portion of calvarial bone to allow unimpeded brain swelling. Hydrogels have the potential to revolutionize TBI treatment by permitting a single-surgical intervention, remaining pliable during brain swelling, and tuned to regenerate bone after swelling has subsided. With this motivation, our goal is to present a pliable material capable of regenerating calvarial bone across a critical size defect. We therefore proposed the use of a methacrylated solubilized decellularized cartilage (MeSDCC) hydrogel encapsulating synthetic osteogenic particles of hydroxyapatite nanofibers, bioglass microparticles, or added rat bone marrow-derived mesenchymal stem cells (rMSCs) for bone regeneration in critical-size rat calvarial defects. Fibrin hydrogels were employed as a control material for the study. MeSDCC hydrogels exhibited sufficient rheological performance for material placement before crosslinking ([Formula: see text] > 500 Pa), and sufficient compressive moduli post-crosslinking (E > 150 kPa). In vitro experiments suggested increased calcium deposition for cells seeded on the MeSDCC material; however, in vivo bone regeneration was minimal in both MeSDCC and fibrin groups, even with colloidal materials or added rMSCs. Minimal bone regeneration in the MeSDCC test groups may potentially be attributed to cartilage solubilization after decellularization, in which material signals may have degraded from enzymatic treatment. Looking to the future, an improvement in the bioactivity of the material will be crucial to the success of bone regeneration strategies for TBI treatment.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton's jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

Extracellular matrix (ECM) "raw materials" such as demineralized bone matrix (DBM) and cartilage matrix have emerged as leading scaffolding materials for osteochondral regeneration owing to their capacity to facilitate progenitor/resident cell recruitment, infiltration, and differentiation without adding growth factors. Scaffolds comprising synthetic polymers are sturdy yet generally lack cues for guiding cell differentiation. We hypothesized that opposing gradients of decellularized cartilage (DCC) and DBM in polymeric microsphere-based scaffolds would provide superior regeneration compared to polymer-only scaffolds in vivo. Poly(D,L-lactic- co -glycolic acid) (PLGA) microsphere-based scaffolds were fabricated, either with opposing gradients of DCC and DBM encapsulated (GRADIENT) or without DCC and DBM (BLANK control), and implanted into rabbit osteochondral defects in medial femoral condyles. After 12 weeks, gross morphological evaluation showed that the repair tissue in about 30% of the implants was either slightly or significantly depressed, hinting toward rapid polymer degradation in scaffolds from both of the groups. Additionally, no differences were observed in gross morphology of the repair tissue between the BLANK and GRADIENT groups. Mechanical testing revealed no significant differences in model parameter values between the two groups. Histological observations demonstrated that the repair tissue in both of the groups was fibrous in nature with the cells demonstrating notable proliferation and matrix deposition activity. No adverse inflammatory response was observed in any of the implants from the two groups. Overall, the results emphasize the need to improve the technology in terms of altering the DBM and DCC concentrations, and tailoring the polymer degradation to these concentrations.

The tissue-engineered heart valve portends a new era in the field of valve replacement. Decellularized heart valves are of great interest as a scaffold for the tissue-engineered heart valve due to their naturally bioactive composition, clinical relevance as a stand-alone implant, and partial recellularization in vivo. However, a significant challenge remains in realizing the tissue-engineered heart valve: assuring consistent recellularization of the entire valve leaflets by phenotypically appropriate cells. Many creative strategies have pursued complete biological valve recellularization; however, identifying the optimal recellularization method, including in situ or in vitro recellularization and chemical and/or mechanical conditioning, has proven difficult. Furthermore, while many studies have focused on individual parameters for increasing valve interstitial recellularization, a general understanding of the interacting dynamics is likely necessary to achieve success. Therefore, the purpose of this review is to explore and compare the various processing strategies used for the decellularization and subsequent recellularization of tissue-engineered heart valves., Competing Interests: Declaration of conflicting interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

To date, many osteochondral regenerative approaches have utilized varied combinations of biocompatible materials and cells to engineer cartilage. Even in cell-based approaches, to date, no study has utilized stem cell aggregates alone for regenerating articular cartilage. Thus, the purpose of this study was to evaluate the performance of a novel stem cell-based aggregate approach in a fibrin carrier to regenerate osteochondral defects in the Sprague-Dawley rat trochlear groove model. Two different densities of rat bone marrow mesenchymal stem cell (rBMSC) aggregates were fabricated by the hanging drop technique. At 8 weeks, the cell aggregates supported the defects and served as a catalyst for neo-cartilage synthesis, and the experimental groups may have been beneficial for bone and cartilage regeneration compared to the fibrin-only control and sham groups, as evidenced by histological assessment. The cell density of rBMSC aggregates may thus directly impact chondrogenesis. The usage of cell aggregates with fibrin as a cell-based technology is a promising and translational new treatment strategy for repair of cartilage defects. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1606-1616, 2017., (© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Microspheres have long been used in drug delivery applications because of their controlled release capabilities. They have increasingly served as the fundamental building block for fabricating scaffolds for regenerative engineering because of their ability to provide a porous network, offer high-resolution control over spatial organization, and deliver growth factors/drugs and/or nanophase materials. Because they provide physicochemical gradients via spatiotemporal release of bioactive factors and nanophase ceramics, microspheres are a desirable tool for engineering complex tissues and biological interfaces. In this review we describe various methods for microsphere fabrication and sintering, and elucidate how these methods influence both micro- and macroscopic scaffold properties, with a special focus on the nature of sintering. Furthermore, we review key applications of microsphere-based scaffolds in regenerating various tissues. We hope to inspire researchers to join a growing community of investigators using microspheres as tissue engineering scaffolds so that their full potential in regenerative engineering may be realized.

Colloidal gels encapsulating natural materials and exhibiting paste-like properties for placement are promising for filling complex geometries in craniofacial bone regeneration applications. Colloidal materials have demonstrated modest clinical outcomes as bone substitutes in orthopedic applications, but limited success in craniofacial applications. As such, development of a novel colloidal gel will fill a void in commercially available products for use in craniofacial reconstruction. One likely application for this technology is cranial reconstruction. Currently, traumatic brain injury (TBI) is often treated with a hemi-craniectomy, a procedure in which half the cranium is removed to allow the injured brain to swell and herniate beyond the enclosed cranial vault. The use of colloidal gels would allow for the design of a pliable material capable of expansion during brain swelling and facilitate cranial bone regeneration alleviating the need for a second surgery to replace the previously removed hemi-cranium. In the current study, colloidal nanoparticles of hydroxyapatite (HAp), demineralized bone matrix (DBM), and decellularized cartilage (DCC) were combined with hyaluronic acid (HA) to form colloidal gels with desirable rheological properties ([Formula: see text] ≥ 100 Pa). BMP-2 and VEGF growth factors were included to assess extracellular matrix (ECM) contribution of DBM and DCC. The HA-HAp (BMP-2) and HA-HAp-DCC group had 89 and 82% higher bone regeneration compared to the sham group, respectively (p < 0.01). Material retention issues observed may be alleviated by implementing chemical crosslinking. Overall, DCC may be a promising material for bone regeneration in general, and colloidal gels may hold significant potential in craniofacial applications.

Decellularized heart valves have great potential as a stand-alone valve replacement or as a scaffold for tissue engineering heart valves. Before decellularized valves can be widely used clinically, regulatory standards require pre-clinical testing in an animal model, often sheep. Numerous decellularization protocols have been applied to both human and ovine valves; however, the ways in which a specific process may affect valves of these species differently have not been reported. In the current study, the comparative effects of decellularization were evaluated for human and ovine aortic valves by measuring mechanical and biochemical properties. Cell removal was equally effective for both species. The initial cell density of the ovine valve leaflets (2036±673cells/mm 2 ) was almost triple the cell density of human leaflets (760±386cells/mm 2 ; p<0.001). Interestingly, post-decellularization ovine leaflets exhibited significant increases in biaxial areal strain (p<0.001) and circumferential peak stretch (p<0.001); however, this effect was not observed in the human counterparts (p>0.10). This species-dependent difference in the effect of decellularization was likely due to the higher initial cellularity in ovine valves, as well as a significant decrease in collagen crosslinking following the decellularization of ovine leaflets that was not observed in the human leaflet. Decellularization also caused a significant decrease in the circumferential relaxation of ovine leaflets (p<0.05), but not human leaflets (p>0.30), which was credited to a greater reduction of glycosaminoglycans in the ovine tissue post-decellularization. These results indicate that an identical decellularization process can have differing species-specific effects on heart valves., Statement of Significance: The decellularized heart valve offers potential as an improved heart valve substitute and as a scaffold for the tissue engineered heart valve; however, the consequences of processing must be fully characterized. To date, the effects of decellularization on donor valves from different species have not been evaluated in such a way that permits direct comparison between species. In this manuscript, we report species-dependent variation in the biochemical and biomechanical properties of human and ovine aortic heart valve leaflets following decellularization. This is of clinical significance, as current regulatory guidelines required pre-clinical use of the ovine model to evaluate candidate heart valve substitutes., (Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Background: Use of decellularized tissues has become popular in tissue engineering applications as the natural extracellular matrix can provide necessary physical cues that help induce the restoration and development of functional tissues. In relation to cochlear tissue engineering, the question of whether decellularized cochlear tissue can act as a scaffold and support the incorporation of exogenous cells has not been addressed. Investigators have explored the composition of the cochlear extracellular matrix and developed multiple strategies for decellularizing a variety of different tissues; however, no one has investigated whether decellularized cochlear tissue can support implantation of exogenous cells., Methods: As a proof-of-concept study, human Wharton's jelly cells were perfused into decellularized cochleae isolated from C57BL/6 mice to determine if human Wharton's jelly cells could implant into decellularized cochlear tissue. Decellularization was verified through scanning electron microscopy. Cocheae were stained with DAPI and immunostained with Myosin VIIa to identify cells. Perfused cochleae were imaged using confocal microscopy., Results: Features of the organ of Corti were clearly identified in the native cochleae when imaged with scanning electron microscopy and confocal microscopy. Acellular structures were identified in decellularized cochleae; however, no cellular structures or lipid membranes were present within the decellularized cochleae when imaged via scanning electron microscopy. Confocal microscopy revealed positive identification and adherence of cells in decellularized cochleae after perfusion with human Wharton's jelly cells. Some cells positively expressed Myosin VIIa after perfusion., Conclusions: Human Wharton's jelly cells are capable of successfully implanting into decellularized cochlear extracellular matrix. The identification of Myosin VIIa expression in human Wharton's jelly cells after implantation into the decellularized cochlear extracellular matrix suggest that components of the cochlear extracellular matrix may be involved in differentiation.

In tissue engineering, an ideal scaffold attracts and supports cells thus providing them with the necessary mechanical support and architecture as they reconstruct new tissue in vitro and in vivo. This manuscript details a novel matrix derived from decellularized Wharton's jelly (WJ) obtained from human umbilical cord for use as a scaffold for tissue engineering application. This decellularized Wharton's jelly matrix (DWJM) contained 0.66 ± 0.12 μg/mg sulfated glycosaminoglycans (GAGs), and was abundant in hyaluronic acid, and completely devoid of cells. Mass spectroscopy revealed the presence of collagen types II, VI and XII, fibronectin-I, and lumican I. When seeded onto DWJM, WJ mesenchymal stem cells (WJMSCs), successfully attached to, and penetrated the porous matrix resulting in a slower rate of cell proliferation. Gene expression analysis of WJ and bone marrow (BM) MSCs cultured on DWJM demonstrated decreased expression of proliferation genes with no clear pattern of differentiation. When this matrix was implanted into a murine calvarial defect model with, green fluorescent protein (GFP) labeled osteocytes, the osteocytes were observed to migrate into the matrix as early as 24 hours. They were also identified in the matrix up to 14 days after transplantation. Together with these findings, we conclude that DWJM can be used as a 3D porous, bioactive and biocompatible scaffold for tissue engineering and regenerative medicine applications.

One of the grand challenges in translational regenerative medicine is the surgical placement of biomaterials. For bone regeneration in particular, malleable and injectable colloidal gelsare frequently designed to exhibit self-assembling and shear-response behavior which facilitates biomaterial placement in tissue defects. The current study demonstrated that by combining native extracellular matrix (ECM) microparticles, i.e., demineralized bone matrix (DBM) and decellularized cartilage (DCC), with hyaluronic acid (HA) and hydroxyapatite (HAP) nanoparticles, a viscoelastic colloidal gel consisting exclusively of natural materials was achieved. Rheological testing of HA-ECM suspensions and HA-HAP-ECM colloidal gels concluded either equivalent or substantially higher storage moduli (G' ≈ 100-10 000 Pa), yield stresses (τ y ≈ 100-1000 Pa), and viscoelastic recoveries (G' recovery ≥ 87%) in comparison with controls formulated without ECM, which indicated a previously unexplored synergy in fluid properties between ECM microparticles and HA-HAP colloidal networks. Notable rheological differences were observed between respective DBM and DCC formulations, specifically in HA-HAP-DBM mixtures, which displayed a mean 3-fold increase in G' and a mean 4-fold increase in τ y from corresponding DCC mixtures. An initial in vitro assessment of these potential tissue fillers as substrates for cell growth revealed that all formulations of HA-ECM and HA-HAP-ECM showed no signs of cytotoxicity and appeared to promote cell viability. Both DBM and DCC colloidal gels represent promising platforms for future studies in bone and cartilage tissue engineering. Overall, the current study identified colloidal gels constructed exclusively of natural materials, with viscoelastic properties that may facilitate surgical placement for a wide variety of therapeutic applications.

Recently, biomaterials-based tissue-engineering strategies, including the use of hydrogels, have offered great promise for repairing articular cartilage. Mechanical failure testing in outcome analyses is of crucial clinical importance to the success of engineered constructs. Interpenetrating networks (IPNs) are gaining more attention, due to their superior mechanical integrity. This study provided a combination testing method of apparent fracture toughness, which was applied to both articular cartilage and hydrogels. The apparent fracture toughnesses of two groups, hydrogels and articular cartilage, were evaluated based on the modified single-edge notch test and ASTM standards on the single-edge notch test and compact tension test. The results demonstrated that the toughness for articular cartilage (348 ± 43 MPa/mm ½ ) was much higher than that for hydrogels. With a toughness value of 10.8 ± 1.4 MPa/mm ½ , IPNs of agarose and poly(ethylene glycol) diacrylate (PEG-DA) looked promising. The IPNs were 1.4 times tougher than PEG-DA alone, although still over an order of magnitude less tough than cartilage. A new method was developed to evaluate hydrogels and cartilage in a manner that enabled a more relevant direct comparison for fracture testing of hydrogels for cartilage tissue engineering. Moreover, a target toughness value for cartilage of using this direct comparison method has been identified (348 ± 43 MPa/mm ½ ), and the toughness discrepancy to be overcome between hydrogels and cartilage has been quantified. Copyright © 2014 John Wiley & Sons, Ltd., Competing Interests: The authors have declared that there is no conflict of interest., (Copyright © 2014 John Wiley & Sons, Ltd.)

The extracellular matrix (ECM) of various tissues possesses the model characteristics that biomaterials for tissue engineering strive to mimic; however, owing to the intricate hierarchical nature of the ECM, it has yet to be fully characterized and synthetically fabricated. Cartilage repair remains a challenge because the intrinsic properties that enable its durability and long-lasting function also impede regeneration. In the last decade, cartilage ECM has emerged as a promising biomaterial for regenerating cartilage, partly because of its potentially chondroinductive nature. As this research area of cartilage matrix-based biomaterials emerged, investigators facing similar challenges consequently developed convergent solutions in constructing robust and bioactive scaffolds. This review discusses the challenges, emerging trends, and future directions of cartilage ECM scaffolds, including a comparison between two different forms of cartilage matrix: decellularized cartilage (DCC) and devitalized cartilage (DVC). To overcome the low permeability of cartilage matrix, physical fragmentation greatly enhances decellularization, although the process itself may reduce the chondroinductivity of fabricated scaffolds. The less complex processing of a scaffold composed of DVC, which has not been decellularized, appears to have translational advantages and potential chondroinductive and mechanical advantages over DCC, without detrimental immunogenicity, to ultimately enhance cartilage repair in a clinically relevant way., (© 2016 New York Academy of Sciences.)

Extracellular matrix materials such as decellularized cartilage (DCC) and chondroitin sulfate (CS) may be attractive chondrogenic materials for cartilage regeneration. The goal of the current study was to investigate the effects of encapsulation of DCC and CS in homogeneous microsphere-based scaffolds, and to test the hypothesis that encapsulation of these extracellular matrix materials would induce chondrogenesis of rat bone marrow stromal cells. Four different types of homogeneous scaffolds were fabricated from microspheres of poly(D,L-lactic-co-glycolic acid): Blank (poly(D,L-lactic-co-glycolic acid) only; negative control), transforming growth factor-β3 encapsulated (positive control), DCC encapsulated, and CS encapsulated. These scaffolds were then seeded with rat bone marrow stromal cells and cultured for 6 weeks. The DCC and CS encapsulation altered the morphological features of the microspheres, resulting in higher porosities in these groups. Moreover, the mechanical properties of the scaffolds were impacted due to differences in the degree of sintering, with the CS group exhibiting the highest compressive modulus. Biochemical evidence suggested a mitogenic effect of DCC and CS encapsulation on rat bone marrow stromal cells with the matrix synthesis boosted primarily by the inherently present extracellular matrix components. An important finding was that the cell seeded CS and DCC groups at week 6 had up to an order of magnitude higher glycosaminoglycan contents than their acellular counterparts. Gene expression results indicated a suppressive effect of DCC and CS encapsulation on rat bone marrow stromal cell chondrogenesis with differences in gene expression patterns existing between the DCC and CS groups. Overall, DCC and CS were easily included in microsphere-based scaffolds; however, there is a requirement to further refine their concentrations to achieve the differentiation profiles we seek in vitro., Competing Interests: Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2016.)

Bioceramic mixtures of tricalcium phosphate (TCP) and hydroxyapatite (HAp) are widely used for bone regeneration because of their excellent cytocompatibility, osteoconduction, and osteoinduction. Therefore, we hypothesized that incorporation of a mixture of TCP and HAp in microsphere-based scaffolds would enhance osteogenesis of rat bone marrow stromal cells (rBMSCs) compared to a positive control of scaffolds with encapsulated bone-morphogenic protein-2 (BMP-2). Poly(D,L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds encapsulating TCP and HAp mixtures in two different ratios (7:3 and 1:1) were fabricated with the same net ceramic content (30 wt%) to evaluate how incorporation of these ceramic mixtures would affect the osteogenesis in rBMSCs. Encapsulation of TCP/HAp mixtures impacted microsphere morphologies and the compressive moduli of the scaffolds. Additionally, TCP/HAp mixtures enhanced the end-point secretion of extracellular matrix components relevant to bone tissue compared to the "blank" (PLGA-only) microsphere-based scaffolds as evidenced by the biochemical, gene expression, histology, and immunohistochemical characterization. Moreover, the TCP/HAp mixture groups even surpassed the BMP-2 positive control group in some instances in terms of matrix synthesis and gene expression. Lastly, gene expression data suggested that the rBMSCs responded differently to different TCP/HAp ratios presented to them. Altogether, it can be concluded that TCP/HAp mixtures stimulated the differentiation of rBMSCs toward an osteoblastic phenotype, and therefore may be beneficial in gradient microsphere-based scaffolds for osteochondral regeneration.

Unlabelled: ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070±150kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance., Statement of Significance: Extracellular matrix (ECM)-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. One such ECM-based material, cartilage ECM, has recently shown potential to be chondroinductive; however, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. Therefore, this work is significant because we were the first to create hydrogels derived entirely from cartilage ECM that had mechanical properties similar to that of native cartilage until hydrogel failure. Furthermore, these hydrogels had a compressive modulus of 1070±150kPa, they were chondroinductive, and they supported extensive matrix synthesis. In the current study, we have shown that these new hydrogels may prove to be a promising biomaterial for cartilage tissue engineering applications., (Copyright © 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Hydrogel precursors are liquid solutions that are prone to leaking from the defect site once implanted in vivo. Therefore, the objective of the current study was to create a hydrogel precursor that exhibited a yield stress. Additionally, devitalized cartilage extracellular matrix (DVC) was mixed with DVC that had been solubilized and methacrylated (MeSDVC) to create hydrogels that were chondroinductive. Precursors composed of 10% MeSDVC or 10% MeSDVC with 10% DVC were first evaluated rheologically, where non-Newtonian behavior was observed in all hydrogel precursors. Rat bone marrow stem cells (rBMSCs) were mixed in the precursor solutions, and the solutions were then crosslinked and cultured in vitro for 6 weeks with and without exposure to human transforming growth factor β3 (TGF-β3). The compressive modulus, gene expression, biochemical content, swelling, and histology of the gels were analyzed. The DVC-containing gels consistently outperformed the MeSDVC-only group in chondrogenic gene expression, especially at 6 weeks, where the relative collagen II expression of the DVC-containing groups with and without TGF-β3 exposure was 40- and 78-fold higher, respectively, than that of MeSDVC alone. Future work will test for chondrogenesis in vivo and overall, these two cartilage-derived components are promising materials for cartilage tissue engineering applications.

"Raw materials," or materials capable of serving both as building blocks and as signals, which are often but not always natural materials, are taking center stage in biomaterials for contemporary regenerative medicine. In osteochondral tissue engineering, a field leveraging the underlying bone to facilitate cartilage regeneration, common raw materials include chondroitin sulfate (CS) for cartilage and β-tricalcium phosphate (TCP) for bone. Building on our previous work with gradient scaffolds based on microspheres, here we delved deeper into the characterization of individual components. In the current study, the release of CS and TCP from poly(D, L-lactic-co-glycolic acid) (PLGA) microsphere-based scaffolds was evaluated over a time period of 4 weeks. Raw material encapsulated groups were compared to 'blank' groups and evaluated for surface topology, molecular weight, and mechanical performance as a function of time. The CS group may have led to increased surface porosity, and the addition of CS improved the mechanical performance of the scaffold. The finding that CS was completely released into the surrounding media by 4 weeks has a significant impact on future in vivo studies, given rapid bioavailability. The addition of TCP seemed to contribute to the rough external appearance of the scaffold. The current study provides an introduction to degradation patterns of homogenous raw material encapsulated scaffolds, providing characterization data to advance the field of microsphere-based scaffolds in tissue engineering., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Tracheal stenosis can become a fatal condition, and current treatments include augmentation of the airway with autologous tissue. A tissue-engineered approach would not require a donor source, while providing an implant that meets both surgeons' and patients' needs. A fibrous, polymeric scaffold organized in gradient bilayers of polycaprolactone (PCL) and poly-lactic-co-glycolic acid (PLGA) with 3D printed structural ring supports, inspired by the native trachea rings, could meet this need. The purpose of the current study was to characterize the tracheal scaffolds with mechanical testing models to determine the design most suitable for maintaining a patent airway. Degradation over 12 weeks revealed that scaffolds with the 3D printed rings had superior properties in tensile and radial compression, with at least a three fold improvement and 8.5-fold improvement, respectively, relative to the other scaffold groups. The ringed scaffolds produced tensile moduli, radial compressive forces, and burst pressures similar to or exceeding physiological forces and native tissue data. Scaffolds with a thicker PCL component had better suture retention and tube flattening recovery properties, with the monolayer of PCL (PCL-only group) exhibiting a 2.3-fold increase in suture retention strength (SRS). Tracheal scaffolds with ring reinforcements have improved mechanical properties, while the fibrous component increased porosity and cell infiltration potential. These scaffolds may be used to treat various trachea defects (patch or circumferential) and have the potential to be employed in other tissue engineering applications.

Purpose: Musculoskeletal tissue engineering has advanced to the stage where it has the capability to engineer temporomandibular joint (TMJ) anatomic components. Unfortunately, there is a paucity of literature identifying specific indications for the use of TMJ tissue engineering solutions. The objective of this study was to establish an initial set of indications and contraindications for the use of engineered tissues for replacement of TMJ anatomic components., Findings: There was consensus among the authors that the management of patients requiring TMJ reconstruction as the result of 1) irreparable condylar trauma, 2) developmental or acquired TMJ pathology in skeletally immature patients, 3) hyperplasia, and 4) documented metal hypersensitivities could be indications for bioengineered condyle and ramus TMJ components. There was consensus that Wilkes stage III internal derangement might be an indication for use of a bioengineered TMJ disc or possibly even a disc-like bioengineered "fossa liner." However, there was some controversy as to whether TMJ arthritic disease (e.g., osteoarthritis) and reconstruction after failed alloplastic devices should be indications. Further research is required to determine whether tissue-engineered TMJ components could be a viable option for such cases. Contraindications for the use of bioengineered TMJ components could include patients with TMJ disorders and multiple failed surgeries, parafunctional oral habits, persistent TMJ infection, TMJ rheumatoid arthritis, and ankylosis unless the underlying pathology can be resolved., Conclusions: Biomedical engineers must appreciate the specific indications that might warrant TMJ bioengineered structures, so that they avoid developing technologies in search of problems that might not exist for patients and clinicians. Instead, they should focus on identifying and understanding the problems that need resolution and then tailor technologies to address those specific situations. The aforementioned indications and contraindications are designed to serve as a guide to the next generation of tissue engineers in their strategic development of technologies to address specific clinical issues., (Copyright © 2016 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-β3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-β3. DVC consistently outperformed all TGF-β3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.

Despite numerous efforts in cartilage regeneration, few products see the light of clinical translation as the commercialization process is opaque, financially demanding, and requires collaboration with people of varied skill sets. The aim of this review is to introduce, to an academic audience, the different paradigms involved in the commercialization of cartilage regeneration technology, elucidate the different hurdles associated with the use of cells and materials in developing new technologies, discuss potential commercialization strategies, and inform the reader about the current trends observed in both the clinical and laboratory setting for establishing clinical trials. Although there are review articles on articular cartilage tissue engineering, independent reports provided by the Food and Drug Administration, and separate review articles on animal models, this is the first review that encompasses all of these facets and is presented in a format favorable to the academic investigator interested in clinical translation from bench to bedside.