Regenerative Engineering of a Biphasic Patient-Fitted Temporomandibular Joint Condylar Prosthesis.

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.

Comparison of multiple synthetic chondroinductive factors in pellet culture against a TGF-ß positive control.

Despite the advances in surgical and cell therapy regenerative techniques for cartilage repair, the challenge is to overcome an inferior fibrocartilage repair tissue. In vitro, TGF-ß1 and TGF-ß3 are the primary growth factors employed to induce chondrogenic differentiation. However, the clinical application of native proteins may present challenges regarding stability, cost, or reproducibility. Therefore, there remains an unmet clinical need for the identification of small chondroinductive synthetic molecules. From the literature, two peptides-CM10 and CK2.1-appear to be promising candidates; however, they have not been directly compared to TGF-ß with human bone marrow-derived stem cells (hBMSCs). Similarly, two promising compounds-kartogenin and SM04690-have been reported in the literature to exhibit chondroinductive potential in vivo and in vitro; however, kartogenin was not directly compared against TGF-ß. In the current study, we evaluated the chondroinductive potential of CM10, CK2.1, kartogenin, and SM04690, and directly compared them to each other and to a TGF-ß3 positive control. Following 21 days of culture, none of the evaluated chondrogenic factors, either individually or even in combinations of two, resulted in a higher gene expression of chondrogenic markers as compared to TGF-ß3. Additionally, no collagen II gene expression was detected except in the TGF-ß3 positive control group. Given that the evaluated factors have confirmed efficacy in the literature, but not in the current study with a positive control, there may be value in the future identification of new chondroinductive factors that are less situation-dependent, with rigorous evaluations of their effect on chondrogenesis using positive controls.

Modulation of Smooth Muscle Cell Phenotype for Translation of Tissue-Engineered Vascular Grafts.

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.

The Ogden model for hydrogels in tissue engineering: Modulus determination with compression to failure.

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, µ0, 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, µ0, 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 µ0 and α for future cartilage tissue engineering studies. The Ogden model was demonstrated to fit the full strain range with high accuracy (R2 = 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.

Comparison of a Thiolated Demineralized Bone Matrix Hydrogel to a Clinical Product Control for Regeneration of Large Sheep Cranial Defects.

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.

High-stiffness, fast-crosslinking, cartilage matrix bioinks.

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.

Independent control of molecular weight, concentration, and stiffness of hyaluronic acid hydrogels.

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 vitroandin vivoto 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.

The Rheology and Printability of Cartilage Matrix-Only Biomaterials.

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.

Automated Decellularization of Musculoskeletal Tissues with High Extracellular Matrix Retention.

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.

Regenerative rehabilitation with conductive biomaterials for spinal cord injury.

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.

Chondroinductive Peptides for Cartilage Regeneration.

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.

Conductive and injectable hyaluronic acid/gelatin/gold nanorod hydrogels for enhanced surgical translation and bioprinting.

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.

Polymer-coated microparticle scaffolds engineered for potential use in musculoskeletal tissue regeneration.

Biomaterials constructed exclusively of sintered microspheres have great potential in tissue engineering scaffold applications, offering the ability to create shape-specific scaffolds with precise controlled release yet to be matched by traditional additive manufacturing methods. The problem is that these microsphere-based scaffolds are limited in their stiffness for applications such as bone regeneration. Our vision to solve this problem was borne from a hierarchical structure perspective, focusing on the individual unit of the structure: the microsphere itself. In a core-shell approach, we envisioned a stiff core to create a stiff microsphere unit, with a polymeric shell that would enable sintering to the other microsphere units. Therefore, the current study provided a comparison of macroscopic biomaterials built on either polymer microspheres or polymer-coated hard glass microspheres. Identical polycaprolactone (PCL) polymer solutions were used to fabricate microspheres and as a thin coating on soda lime glass microspheres (hard phase). The materials were characterized as loose particles and as scaffolds via scanning electron microscopy, thermogravimetry, differential scanning calorimetry, Raman spectroscopy, mechanical testing, and a live/dead analysis with human umbilical cord-derived Wharton's jelly cells. The elastic modulus of the scaffolds with the thinly coated hard phase was about five times higher with glass microspheres (up to about 25 MPa) than pure polymer microspheres, while retaining the structure, cell adhesion, and chemical properties of the PCL polymer. This proof-of-concept study demonstrated the ability to achieve at least a five-fold increase in macroscopic stiffness via altering the core microsphere units with a core-shell approach.

Unrepaired decompressive craniectomy worsens motor performance in a rat traumatic brain injury model.

Decompressive craniectomy (DC) is often required to manage rising intracranial pressure after traumatic brain injury (TBI). Syndrome of the trephine (SoT) is a reversible neurologic condition that often occurs following DC as a result of the unrepaired skull. The purpose of the present study is to characterize neurological impairment following TBI in rats with an unrepaired craniectomy versus rats with a closed cranium. Long Evans male rats received a controlled cortical impact (CCI) over the caudal forelimb area (CFA) of the motor cortex. Immediately after CCI, rats received either a hemi-craniectomy (TBI Open Skull Group) or an immediate acrylic cranioplasty restoring cranial anatomy (TBI Closed Skull Group). Motor performance was assessed on a skilled reaching task on post-CCI weeks 1-4, 8, 12, and 16. Three weeks after the CCI injury, the TBI Closed Skull Group demonstrated improved motor performance compared to TBI Open Skull Group. The TBI Closed Skull Group continued to perform better than the TBI Open Skull Group throughout weeks 4, 8, 12 and 16. The protracted recovery of CFA motor performance demonstrated in rats with unrepaired skulls following TBI suggests this model may be beneficial for testing new therapeutic approaches to prevent SoT.

Manifestations of Apprehension and Anxiety in a Sprague Dawley Cranial Defect Model.

BACKGROUND: Syndrome of the trephined is a neurologic condition that commonly arises in patients who undergo craniectomy and have a prolonged cranial defect. Symptoms of this condition include headache, difficulties concentrating, diminished fine motor/dexterity skills, mood changes, and anxiety/apprehension. The authors hypothesize that an animal model demonstrating anxiety/apprehension in rats who undergo craniectomy is feasible utilizing standardized animal behavioral testing. METHODS: Sprague Dawley rats were the stratified to 1 of 2 groups for comparison of neurobehavioral outcomes. Group #1 (closed cranial group) had their cranial trephination immediately closed with acrylic to restore normal cranial anatomy and Group #2 (open cranial group) had their cranial trephination enlarged to represent a decompressive hemicraniectomy immediately. Anxiety/apprehension was studied using a standardized rodent open field test. Statistical comparison of differences among the 2 groups was performed. RESULTS: Ten rats were studied with 5 rats in each group. Standard rodent open field testing of anxiety demonstrated no difference among the 2 groups at 1 week. Rats in the "Open cranial group" demonstrated progressively more anxiety over the following 3-month period. Rats in the "Open cranial group" demonstrated increasing anxiety levels as compared with rats in the "Closed cranial group." At week 16, the "Open cranial group" anxiety levels were significantly greater than week 4 (t = 2.24, P = 0.04) demonstrating a significant linear trend over time (R = 0.99; P = 0.002). The "Closed cranial group" did not show this trend (R = 07; P = 0.74). CONCLUSION: Our study demonstrates that anxiety and apprehension are more prevalent in rats with an open, prolonged cranial defect in comparison to those with a closed cranium. This correlates with similar finds in humans with syndrome of the trephined.

Standardization of Microcomputed Tomography for Tracheal Tissue Engineering Analysis.

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.

Assessing nanoparticle colloidal stability with single-particle inductively coupled plasma mass spectrometry (SP-ICP-MS).

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.

Thiolated bone and tendon tissue particles covalently bound in hydrogels for in vivo calvarial bone regeneration.

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.

Temporomandibular Joint Bioengineering Conference: Working Together Toward Improving Clinical Outcomes.

The sixth temporomandibular joint (TMJ) Bioengineering Conference (TMJBC) was held on June 14-15 2018, in Redondo Beach, California, 12 years after the first TMJBC. Speakers gave 30 presentations and came from the United States, Europe, Asia, and Australia. The goal of the conference has remained to foster a continuing forum for bioengineers, scientists, and surgeons and veterinarians to advance technology related to TMJ disorders. These collective multidisciplinary interactions over the past decade have made large strides in moving the field of TMJ research forward. Over the past 12 years, in vivo approaches for tissue engineering have emerged, along with a wide variety of degeneration models, as well as with models occurring in nature. Furthermore, biomechanical tools have become more sensitive and new biologic interventions for disease are being developed. Clinical directives have evolved for specific diagnoses, along with patient-specific biological and immunological responses to TMJ replacement devices alloplastic and/or bioengineered devices. The sixth TMJBC heralded many opportunities for funding agencies to advance the field: (1) initiatives on TMJ that go beyond pain research, (2) more training grants focused on graduate students and fellows, (3) partnership funding with government agencies to translate TMJ solutions, and (4) the recruitment of a critical mass of TMJ experts to participate on grant review panels. The TMJ research community continues to grow and has become a pillar of dental and craniofacial research, and together we share the unified vision to ultimately improve diagnoses and treatment outcomes in patients affected by TMJ disorders.

Biodegradable electrospun patch containing cell adhesion or antimicrobial compounds for trachea repair in vivo.

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.