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.

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.

Flow Behavior Prior to Crosslinking: The Need for Precursor Rheology for Placement of Hydrogels in Medical Applications and for 3D Bioprinting.

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.

Microsphere-Based Scaffolds in Regenerative Engineering.

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.

Hyaluronic-Acid-Hydroxyapatite Colloidal Gels Combined with Micronized Native ECM as Potential Bone Defect Fillers.

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 (τy ≈ 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 τy 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.

Potential Indications for Tissue Engineering in Temporomandibular Joint Surgery.

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.

Microsphere-based scaffolds encapsulating tricalcium phosphate and hydroxyapatite for bone regeneration.

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.

The effect of extended passaging on the phenotype and osteogenic potential of human umbilical cord mesenchymal stem cells.

Retaining biological characteristics in the extended passaging is crucial for human umbilical cord mesenchymal stem cells (hUCMSCs) in tissue engineering. We aimed to assess morphology, viability, MSC marker expression, and osteogenic activity of hUCSMCs after extended passaging. Passages 4 (P4) and 16 (P16) hUCMSCs displayed similar morphology and viability. The flow cytometry results showed that CD73, CD90, and CD105 were highly expressed at P1-P16. CD166 expression decreased progressively from 90 % at P2 to 61.5 % at P5 (p < 0.05), followed by stable expression through P16. Results from calcium deposition alkaline phosphatase activity and RT-PCR assay showed that both P4 and P16 hUCMSCs differentiated down an osteogenic lineage, with no significant difference in osteogenic capacity (p < 0.05). High-passage UMCSCs maintained stable expression of MSC CD markers as well as stable osteogenic activity. hUCMSCs may thus be suitable for tissue engineering and regenerative medicine applications.

The potential of encapsulating "raw materials" in 3D osteochondral gradient scaffolds.

Scaffolds with continuous gradients in material composition and bioactive signals enable a smooth transition of properties at the interface. Components like chondroitin sulfate (CS) and bioactive glass (BG) in 3D scaffolds may serve as "raw materials" for synthesis of new extracellular matrix (ECM), and may have the potential to completely or partially replace expensive growth factors. We hypothesized that scaffolds with gradients of ECM components would enable superior performance of engineered constructs. Raw material encapsulation altered the appearance, structure, porosity, and degradation of the scaffolds. They allowed the scaffolds to better retain their 3D structure during culture and provided a buffering effect to the cells in culture. Following seeding of rat mesenchymal stem cells, there were several instances where glycosaminoglycan (GAG), collagen, or calcium contents were higher with the scaffolds containing raw materials (CS or BG) than with those containing transforming growth factor (TGF)-ß3 or bone morphogenetic protein (BMP)-2. It was also noteworthy that a combination of both CS and TGF-ß3 increased the secretion of collagen type II. Moreover, cells seeded in scaffolds containing opposing gradients of CS/TGF-ß3 and BG/BMP-2 produced clear regional variations in the secretion of tissue-specific ECM. The study demonstrated raw materials have the potential to create a favorable microenvironment for cells; they can significantly enhance the synthesis of certain extracellular matrix (ECM) components when compared to expensive growth factors; either alone or in combination with growth factors they can enhance the secretion of tissue specific matrix proteins. Raw materials are promising candidates that can be used to either replace or be used in combination with growth factors. Success with raw materials in lieu of growth factors could have profound implications in terms of lower cost and faster regulatory approval for more rapid translation of regenerative medicine products to the clinic.

Mapping glycosaminoglycan-hydroxyapatite colloidal gels as potential tissue defect fillers.

Malleable biomaterials such as Herschel-Bulkley (H-B) fluids possess shear responsive rheological properties and are capable of self-assembly and viscoelastic recovery following mechanical disruption (e.g., surgical placement via injection or spreading). This study demonstrated that the addition of moderate molecular weight glycosaminoglycans (GAGs) such as chondroitin sulfate (CS) (Mw = 15-30 kDa) and hyaluronic acid (HA) (Mw = 20-41 kDa) can be used to modify several rheological properties including consistency index (K), flow-behavior index (n), and yield stress (τy) of submicrometer hydroxyapatite (HAP) (Davg ≤ 200 nm) colloidal gels. GAG-HAP colloidal mixtures exhibited substantial polymer-particle synergism, likely due to "bridging" flocculation, which led to a synergistic increase in consistency index (KGAG-HAP ≥ KGAG + KHAP) without compromising shear-thinning behavior (n < 1) of the gel. In addition, GAG-HAP colloids containing high concentrations of HAP (60-80% w/v) exhibited substantial yield stress (τy ≥ 100 Pa) and viscoelastic recovery properties (G'recovery ≥ 64%). While rheological differences were observed between CS-HAP and HA-HAP colloidal gels, both CS and HA represent feasible options for future studies involving bone defect filling. Overall, this study identified mixture regions where rheological properties in CS-HAP and HA-HAP colloidal gels aligned with desired properties to facilitate surgical placement in non-load-bearing tissue-filling applications such as calvarial defects.

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.

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.

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.

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.

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.

Using chondroitin sulfate to improve the viability and biosynthesis of chondrocytes encapsulated in interpenetrating network (IPN) hydrogels of agarose and poly(ethylene glycol) diacrylate.

We recently introduced agarose-poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN) hydrogels to cartilage tissue engineering that were able to encapsulate viable cells and provide a significant improvement in mechanical performance relative to its two constituent hydrogels. The goal of the current study was to develop a novel synthesis protocol to incorporate methacrylated chondroitin sulfate (MCS) into the IPN design hypothesized to improve cell viability and biosynthesis. The IPN was formed by encapsulating porcine chondrocytes in agarose, soaking the construct in a solution of 1:10 MCS:PEGDA, which was then photopolymerized to form a copolymer network as the second network. The IPN with incorporated CS (CS-IPN) (~0.5 wt%) resulted in a 4- to 5-fold increase in the compressive elastic modulus relative to either the PEGDA or agarose gels. After 6 weeks of in vitro culture, more than 50% of the encapsulated chondrocytes remained viable within the CS-modified IPN, in contrast to 35% viability observed in the unmodified. At week 6, the CS-IPN had significantly higher normalized GAG contents (347 ± 34 µg/µg) than unmodified IPNs (158 ± 27 µg/µg, P < 0.05). Overall, the approach of incorporating biopolymers such as CS from native tissue may provide favorable micro-environment and beneficial signals to cells to enhance their overall performance in IPNs.

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.

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.