Effective Light Directed Assembly of Building Blocks with Microscale Control.

Light-directed forces have been widely used to pattern micro/nanoscale objects with precise control, forming functional assemblies. However, a substantial laser intensity is required to generate sufficient optical gradient forces to move a small object in a certain direction, causing limited throughput for applications. A high-throughput light-directed assembly is demonstrated as a printing technology by introducing gold nanorods to induce thermal convection flows that move microparticles (diameter = 40 µm to several hundreds of micrometers) to specific light-guided locations, forming desired patterns. With the advantage of effective light-directed assembly, the microfluidic-fabricated monodispersed biocompatible microparticles are used as building blocks to construct a structured assembly (≈10 cm scale) in ≈2 min. The control with microscale precision is approached by changing the size of the laser light spot. After crosslinking assembly of building blocks, a novel soft material with wanted pattern is approached. To demonstrate its application, the mesenchymal stem-cell-seeded hydrogel microparticles are prepared as functional building blocks to construct scaffold-free tissues with desired structures. This light-directed fabrication method can be applied to integrate different building units, enabling the bottom-up formation of materials with precise control over their internal structure for bioprinting, tissue engineering, and advanced manufacturing.

Stem cell-derived cell-sheets for connective tissue engineering.

Cell-sheet technology involves the recovery of cells with its secreted ECM and cell-cell junctions intact, and thereby harvesting them in a single contiguous layer. Temperature changes coupled with a thermoresponsive polymer grafted culture plate surface are typically used to induce detachment of this cell-matrix layer by controlling the hydrophobicity and hydrophilicity properties of the culture surface. This review article details the genesis and development of this technique as a critical tissue-engineering tool, with a comprehensive discussion on connective tissue applications. This includes applications in the myocardial, vascular, cartilage, bone, tendon/ligament, and periodontal areas among others discussed. In particular, further focus will be given to the use of stem cells-derived cell-sheets, such as those involving bone marrow-derived and adipose tissue-derived mesenchymal stem cells. In addition, some of the associated challenges faced by approaches using stem cells-derived cell-sheets will also be discussed. Finally, recent advances pertaining to technologies forming, detaching, and manipulating cell-sheets will be covered in view of the potential impact they will have on shaping the way cell-sheet technology will be utilized in the future as a tissue-engineering technique.

Silk fibroin-based complex particles with bioactive encrustation for bone morphogenetic protein 2 delivery.

Application of bone morphogenetic protein 2 (BMP-2) currently faces its challenges, and its efficacy of delivery has to be improved. The proper dosage of the powerful bioactive molecule is still under discussion and needs to be investigated further. In this work, pure silk fibroin particles and particles with calcium carbonate encrustation (complex particles) are designed, developed, and functionalized by BMP-2. These are used to deliver the bioactive molecule to mesenchymal stem cells (MSCs) to induce osteogenic differentiation. Results are compared with those of control groups of BMP-2 carriers under the same condition. Silk fibroin-based particles with size and component variations are prepared by self-assembly, desolvation, and soft template formation to improve BMP-2 loading efficiency. Results show that the particles significantly enhance osteogenic differentiation of MSCs, which is evident in the high ALP enzyme activity as well as the increased level of expression of osteogenic genes. Specifically, the combination of calcium compound and BMP-2 in the silk fibroin-calcium carbonate complex particles synergistically enhances osteogenesis. Release tests and mathematical modeling are applied to describe BMP-2 dissolution profiles, and the release mechanism is based on diffusion and polymer chain relaxation. In summary, the particles show high efficacies of BMP-2 delivery, and introduction of the complex particle can progressively enhance osteogenesis.

The effect of chondroitin sulfate concentration and matrix stiffness on chondrogenic differentiation of mesenchymal stem cells.

Chondroitin sulfate (CS), a glycosaminoglycan of native cartilage, has shown its potential in promoting chondrogenesis of mesenchymal stem cells (MSCs), whereas the effect of matrix stiffness in a CS-containing 3D environment on chondrogenesis is still poorly understood. Herein, this study aimed at assessing the effect of CS concentration and stiffness of CS-containing hydrogels on the chondrogenesis of MSCs. Hydrogels composed of 6% (w/v) gelatin methacryloyl (GelMA) and three concentrations, i.e., 4%, 6%, or 10% (w/v), of methacrylated chondroitin sulfate (CSMA) were prepared. The hydrogels of each composition were prepared with two stiffness values (33.36 ± 8.25 kPa vs. 8.42 ± 2.83 kPa). Physical characterization showed similar microporous structures among the six groups, higher swelling ratios and faster degradation in the soft hydrogel groups. MSCs were encapsulated in the six groups of hydrogels and they underwent 28-day chondrogenic differentiation. The cell viability in each group on day 1 was similar and most cells exhibited a round shape without spreading. Afterwards, cellular protrusions in soft hydrogels remained filopodium-like from day 14 to day 28, while most protrusions were lamellipodium-like in stiff hydrogels on day 14 and then transformed into a spherical shape on day 28. The expression of chondrogenic markers analysed by real-time qPCR and immunohistochemical staining demonstrated that the optimal CS concentration for chondrogenesis was 6% (w/v) regardless of the stiffness of hydrogels. In addition, with the same CSMA concentration, the trend was observed that the stiff hydrogels supported superior chondrogenesis of MSCs compared to the soft hydrogel. To summarize, this study presents an advancement in the optimization of CSMA concentration and stiffness of hydrogels for chondrogenesis. In the CSMA/GelMA hydrogel, 6% (w/v) CSMA with an initial Young's modulus around 33 kPa was recommended for cartilage tissue engineering.

SIRIUS, Ultra-Scintillating Upconversion Breast Implant for Remote Orthotopic Photodynamic Therapy.

Present day strategies for delivery of wireless photodynamic therapy (PDT) to deep-seated targets are limited by the inadequacy of irradiance and insufficient therapeutic depth. Here we report the design and preclinical validation of a flexible wireless upconversion nanoparticle (UCNP) implant (SIRIUS) that is capable of large field, high intensity illumination for PDT of deep-seated tumors. The implant achieves this by incorporating submicrometer core-shell-shell NaYF4 UCNPs into its design, which significantly enhances upconversion efficiency and mitigates light loss from surface quenching. We demonstrate the efficacy of SIRIUS UCNP implant mediated PDT in preclinical breast cancer disease models. In our in vitro experiments, SIRIUS directed 5-Aminolevulinic Acid (5-ALA) based wireless PDT leads to significant reactive oxygen species (ROS) generation and tumor apoptosis in hormonal receptor+/HER2+ (MCF7) and triple-negative (MDA-MB-231) breast cancer cell lines. In our in vivo rodent model, SIRIUS-driven PDT is shown to be significant in regressing tumors when applied to orthotopically inoculated breast tumors. Following successful preclinical validation, we also describe a clinical prototype of UCNP breast implant with potential dual cosmetic and onco-therapeutic functions. SIRIUS is an upconversion breast implant for wireless PDT that fulfils all the design prerequisites necessary for seamless clinical translation.

In vitro ligament-bone interface regeneration using a trilineage coculture system on a hybrid silk scaffold.

The ligament-bone interface is a complex structure that comprises ligament, fibrocartilage, and bone. We hypothesize that mesenchymal stem cells cocultured in between ligament and bone cells, on a hybrid silk scaffold with sections suitable for each cell type, would differentiate into fibrocartilage. The section of scaffold for osteoblast seeding was coated with hydroxyapatite. A trilineage coculture system (osteoblasts-BMSCs-fibroblasts) on a hybrid silk scaffold was established. RT-PCR results and immunohistochemistry results demonstrated that BMSCs cocultured between fibroblasts and osteoblasts had differentiated into the fibrocartilaginous lineage. The morphological change was also observed by SEM observation. A gradual transition from the uncalcified to the calcified region was formed in the cocultured BMSCs from the region that directly interacted with fibroblasts to the region that directly interacted with osteoblasts. The role of transforming growth factor ß3 (TGF-ß3) in this trilineage coculture model was also investigated by supplementing the coculture system with 10 ng/mL TGF-ß3. The TGF-treated group showed similar results of fibrocartilaginous differentiation of BMSCs with coculture group without TGF-ß3 supplement. However, no calcium deposition was found in the cocultured BMSCs in the TGF-treated group. This may indicate TGF-ß3 delayed the mineralization process of chondrocytes.

Silk Fibroin as a Bioink - A Thematic Review of Functionalization Strategies for Bioprinting Applications.

Bioprinting is an emerging tissue engineering technique that has attracted the attention of researchers around the world, for its ability to create tissue constructs that recapitulate physiological function. While the technique has been receiving hype, there are still limitations to the use of bioprinting in practical applications, much of which is due to inappropriate bioink design that is unable to recapitulate complex tissue architecture. Silk fibroin (SF) is an exciting and promising bioink candidate that has been increasingly popular in bioprinting applications because of its processability, biodegradability, and biocompatibility properties. However, due to its lack of optimum gelation properties, functionalization strategies need to be employed so that SF can be effectively used in bioprinting applications. These functionalization strategies are processing methods which allow SF to be compatible with specific bioprinting techniques. Previous literature reviews of SF as a bioink mainly focus on discussing different methods to functionalize SF as a bioink, while a comprehensive review on categorizing SF functional methods according to their potential applications is missing. This paper seeks to discuss and compartmentalize the different strategies used to functionalize SF for bioprinting and categorize the strategies for each bioprinting method (namely, inkjet, extrusion, and light-based bioprinting). By compartmentalizing the various strategies for each printing method, the paper illustrates how each strategy is better suited for a target tissue application. The paper will also discuss applications of SF bioinks in regenerating various tissue types and the challenges and future trends that SF can take in its role as a bioink material.

Stretchable and Self-Adhesive PEDOT:PSS Blend with High Sweat Tolerance as Conformal Biopotential Dry Electrodes.

Dry epidermal electrodes that can always form conformal contact with skin can be used for continuous long-term biopotential monitoring, which can provide vital information for disease diagnosis and rehabilitation. But, this application has been limited by the poor contact of dry electrodes on wet skin. Herein, we report a biocompatible fully organic dry electrode that can form conformal contact with both dry and wet skin even during physical movement. The dry electrodes are prepared by drop casting an aqueous solution consisting of poly(3,4-ethylenedioxythiophene):polystyrenesulfonate (PEDOT:PSS), poly(vinyl alcohol) (PVA), tannic acid (TA), and ethylene glycol (EG). The electrodes can exhibit a conductivity of 122 S cm-1 and a mechanical stretchability of 54%. Moreover, they are self-adhesive to not only dry skin but also wet skin. As a result, they can exhibit a lower contact impedance to skin than commercial Ag/AgCl gel electrodes on both dry and sweat skins. They can be used as dry epidermal electrodes to accurately detect biopotential signals including electrocardiogram (ECG) and electromyogram (EMG) on both dry and wet skins for the users at rest or during physical movement. This is the first time to demonstrate dry epidermal electrodes self-adhesive to wet skin for accurate biopotential detection.

Bone marrow mesenchymal stem cells with low dose bone morphogenetic protein 2 enhances scaffold-based spinal fusion in a porcine model.

High doses bone morphogenetic protein 2 (BMP-2) have resulted in a series of complications in spinal fusion. We previously established a polyelectrolyte complex (PEC) carrier system that reduces the therapeutic dose of BMP-2 in both rodent and porcine spinal fusion models. This study aimed to evaluate the safety and efficacy of the combination of bone marrow mesenchymal stem cells (BMSCs) and low dose BMP-2 delivered by PEC for bone regeneration in a porcine model of anterior lumbar interbody spinal fusion (ALIF) application. Six Yorkshire pigs underwent a tri-segmental (L2/L3; L3/L4; L4/L5) ALIF in four groups, namely: (a) BMSCs + 25 µg BMP-2/PEC (n = 9), (b) 25 µg BMP-2/PEC (n = 3), (c) BMSCs (n = 3), and (d) 50 µg BMP-2/absorbable collagen sponge (n = 3). Fusion outcomes were evaluated by radiography, biomechanical testing, and histological analysis after 12 weeks. Mean radiographic scores at 12 weeks were 2.7, 2.0, 1.0, and 1.0 for Groups 1 to 4, respectively. µ-CT scanning, biomechanical evaluation, and histological analysis demonstrated solid fusion and successful bone regeneration in Group 1. In contrast, Group 2 showed inferior quality and slow rate of fusion, and Groups 3 and 4 failed to fuse any of the interbody spaces. There was no obvious evidence of seroma formation, implant rejection, or any other complications in all groups. The results suggest that the combination of BMSCs and low dose BMP-2/PEC could further lower down the effective dose of the BMP-2 and be used as a bone graft substitute in the large animal ALIF model.

Bioactive nanofibers for fibroblastic differentiation of mesenchymal precursor cells for ligament/tendon tissue engineering applications.

Mesenchymal stem cells and precursor cells are ideal candidates for tendon and ligament tissue engineering; however, for the stem cell-based approach to succeed, these cells would be required to proliferate and differentiate into tendon/ligament fibroblasts on the tissue engineering scaffold. Among the various fiber-based scaffolds that have been used in tendon/ligament tissue engineering, hybrid fibrous scaffolds comprising both microfibers and nanofibers have been recently shown to be particularly promising. With the nanofibrous coating presenting a biomimetic surface, the scaffolds can also potentially mimic the natural extracellular matrix in function by acting as a depot for sustained release of growth factors. In this study, we demonstrate that basic fibroblast growth factor (bFGF) could be successfully incorporated, randomly dispersed within blend-electrospun nanofibers and released in a bioactive form over 1 week. The released bioactive bFGF activated tyrosine phosphorylation signaling within seeded BMSCs. The bFGF-releasing nanofibrous scaffolds facilitated BMSC proliferation, upregulated gene expression of tendon/ligament-specific ECM proteins, increased production and deposition of collagen and tenascin-C, reduced multipotency of the BMSCs and induced tendon/ligament-like fibroblastic differentiation, indicating their potential in tendon/ligament tissue engineering applications.

Correlation of axial impact forces with knee joint forces and kinematics during simulated ski-landing.

Anterior cruciate ligament (ACL) rupture, during ski-landing, is caused by excessive knee joint forces and kinematics, like anterior tibial translation, internal tibial rotation, and valgus rotation. It is not well understood how these forces/kinematics are directly related to ski-landing impact. In the present study, we applied simulated ski-landing impact to knee specimens, and examined joint force/kinematic responses and their correlations with impact force. Ten human cadaveric knees were subjected to axial impact loading at 70° of flexion to simulate ski-landing impact. Impact was repeated with incremental magnitude until ACL failure. Axial impact forces, anterior-posterior and medial-lateral tibial forces were measured using a tri-axial load cell. Anterior-posterior tibial translation, internal-external tibial rotation, and valgus-varus rotation were determined using a motion-capture system. We found positive correlations of axial impact force with anterior tibial force, medial tibial force, anterior tibial translation, internal tibial rotation, and valgus joint rotation. Axial impact forces were more strongly correlated with anterior tibial forces (R(2) = 0.937 ± 0.050), anterior tibial translation (R(2) = 0.916 ± 0.059), and internal tibial rotation (R(2) = 0.831 ± 0.141) than medial tibial force (R(2) = 0.677 ± 0.193) and valgus joint rotation (R(2) = 0.630+0.271). During ski-landing, these joint forces/kinematics can synergistically act to increase ACL injury risk, whereby the failure mechanism would be dominated by anterior tibial forces, anterior tibial translation, and internal tibial rotation.

Pore size modulates in vitro osteogenesis of bone marrow mesenchymal stem cells in fibronectin/gelatin coated silk fibroin scaffolds.

Porous scaffolds have been widely used for bone tissue engineering (BTE), and the pore structure of scaffolds plays an important role in osteogenesis. Silk fibroin (SF) is a favorable biomaterial for BTE due to its excellent mechanical property, biocompatibility, and biodegradation, but the lack of cell attachment sites in SF chemical structure resulted in poor cell-material interactions. In this study, SF scaffolds were coated with fibronectin/gelatin (Fn/G) to improve cell adhesion. Furthermore, the effect of pore size in Fn/G coated SF scaffolds on osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were investigated in vitro. Scaffolds with average pore diameters of 384.52, 275.23, and 173.8 µm were prepared by salt leaching method, labelled as Large, Medium, and Small group. Porcine BMSCs were seeded on scaffolds and cultured in osteogenic medium for 21 days to evaluate cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, gene expression of osteogenic markers, and histological performance. The results showed Fn/G coating effectively improved cell adhesion on SF scaffolds. Cell metabolic rate in each group increased significantly with time, but there was no statistical difference at each time point among the three groups. On day 21, ALP/DNA and calcium/DNA in the Small group were significantly higher than those in the Large group. Among the three pore sizes, the Small group showed higher mRNA expression of COl I on day 7, OPN on day 14, and OCN on day 21. Immunohistochemical staining on day 21 showed that Col I and OCN in Small group were more highly expressed. In conclusion, the Fn/G coated SF scaffolds with a mean pore diameter of 173.8 µm was optimal for osteogenic differentiation of BMSC in vitro.

Conductive polypyrrole-encapsulated silk fibroin fibers for cardiac tissue engineering.

Polypyrrole (PPy) has been utilized in smart scaffolds to improve the functionality of the engineered cardiac tissue. Compared to the commonly used aqueous coating, here, PPy was blended into silk fibroin (SF) solution to electrospin conductive PPy-encapsulated SF nanofibers. Combinations of various SF concentrations (5%, 7%, and 12%) and different PPy-to-SF ratios (15:85, 30:70, and 40:60) were compared. PPy reduced the fiber diameter (0.431 ± 0.060 µm), better-mimicking the myocardium fibrils. Conductive mats with 7% SF showed the closest mechanical properties (1.437 ± 0.044 MPa) to the native myocardium; meanwhile, a PPy-to-SF ratio of 15:85 exhibited sufficient electrical conductivity for cardiomyocytes (CMs). In vitro studies using three different types of CM demonstrated that the hybrid mats support CM contraction. Primary neonatal rat CMs on the mat with a PPy-to-SF ratio of 15:85 were elongated and orientated anisotropically with locally organized sarcomeric striations. By contrast, human-induced pluripotent stem cell derived-CMs on the mat with a PPy-to-SF ratio of 30:70 exhibited the strongest contractions. Contraction synchrony was further improved by external stimulation. Taken together, these findings indicated the great potential of the PPy-encapsulated SF electrospun mat for cardiac tissue engineering.

Osteochondral tissue engineering: Perspectives for clinical application and preclinical development.

The treatment of osteochondral defects (OCD) remains challenging. Among currently available surgical treatments for OCDs, scaffold-based treatments are promising to regenerate the osteochondral unit. However, there is still no consensus regarding the clinical effectiveness of these scaffold-based therapies for OCDs. Previous reviews have described the gradient physiological characteristics of osteochondral tissue and gradient scaffold design for OCD, tissue engineering strategies, biomaterials, and fabrication technologies. However, the discussion on bridging the gap between the clinical need and preclinical research is still limited, on which we focus in the present review, providing an insight into what is currently lacking in tissue engineering methods that failed to yield satisfactory outcomes, and what is needed to further improve these techniques. Currently available surgical treatments for OCDs are firstly summarized, followed by a comprehensive review on experimental animal studies in recent 5 years on osteochondral tissue engineering. The review will then conclude with what is currently lacking in these animal studies and the recommendations that would help enlighten the community in developing more clinically relevant implants. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This review is attempting to summarize the lessons from clinical and preclinical failures, providing an insight into what is currently lacking in TE methods that failed to yield satisfactory outcomes, and what is needed to further improve these implants.

Fabrication of 3D-Printed Ceramic Structures for Portable Solar Desalination Devices.

This paper proposes the fabrication process of the first fully 3D-printed ceramic core structures for portable solar desalination devices optimized to tackle water scarcity from an energy and sustainability perspective. Robocasting, a 3D printing technique, is utilized to fabricate a fully ceramic structure of an integrated solar absorber/thermal insulator/water transporter based on the two-layered structure of modified graphene on silica (MG@Silica) and the porous silica structure. Robocasting has demonstrated its flexibility in tailoring structural designs, combining nanopores and microchannels that exhibit uniform water transport delivery and thermal insulation. This portable device can be used immediately to collect fresh drinking water without an additional setup. It possesses a water evaporation rate of 2.4 kg m-2 h-1 with a drinking water production capacity of 0.5 L m-2 h-1. This novel device has shown excellent ion rejection ability, with the collected water meeting the World Health Organization (WHO) drinking water standards.

Bio-electrospraying: A potentially safe technique for delivering progenitor cells.

Bio-electrospraying is fast becoming an attractive tool for in situ cell delivery into scaffolds for tissue engineering applications, with several cell types been successfully electrosprayed. Bone marrow derived mesenchymal progenitor/stem cells (BMSC), which are an important cell source for tissue engineering, have not been explored in detail and the effect of electrospraying on their "stemness" is not known. This study therefore investigates the effects of electrospraying on BMSC viability, proliferation, and multilineage differentiation potential. Electrospraying a BMSC suspension at flow rate of 6 mL/h and voltages of 7.5-15 kV could successfully generate a continuous, stable and linearly directed electrospray of cells. Morphological observation, trypan blue tests and alamar blue based metabolic assays revealed about 88% of these electrosprayed cells were viable, and proliferated at rates similar to native BMSCs. However, at higher voltages, electrospraying became unstable and reduced cell viability, possibly due to electrical or thermal damage to the cells. BMSCs electrosprayed at 7.5 kV also retained their multipotency and could be successfully differentiated into adipogenic, chondrogenic, and osteogenic lineages, demonstrating similar morphology and gene expression levels as induced native BMSCs. These results indicate that bio-electrospraying could be safely used as a progenitor/stem cell delivery technique for tissue engineering and regenerative medicine applications.

Polypyrrole-Incorporated Conducting Constructs for Tissue Engineering Applications: A Review.

Conductive polymers have recently attracted interest in biomedical applications because of their excellent intrinsic electrical conductivity and satisfactory biocompatibility. Polypyrrole (PPy) is one of the most popular among these conductive polymers due to its high conductivity under physiological conditions, and it can be chemically modified to allow biomolecules conjugation. PPy has been used in fabricating biocompatible stimulus-responsive scaffolds for tissue engineering applications, especially for repair and regeneration of electroactive tissues, such as the bone, neuron, and heart. This review provides a comprehensive overview of the basic properties and synthesis methods of PPy, as well as a summary of the materials that have been integrated with PPy. These composite scaffolds are comparatively evaluated with regard to their mechanical properties, biocompatibility, and usage in tissue engineering.

Improving the handling properties and long-term stability of polyelectrolyte complex by freeze-drying technique for low-dose bone morphogenetic protein 2 delivery.

A variety of controlled release carriers for bone morphogenetic protein 2 (BMP-2) delivery have been developed and tested in animal models. An alginate-based polyelectrolyte complex (PEC) for controlled release of low-dose BMP-2 has shown promising results in preclinical research. However, the poor handling properties and long-term stability of PEC need to be improved for translational applications. This study aimed to address these limitations of alginate-based PEC by employing a freeze-drying technique. The size and structure of freeze-dried PEC (FD-PEC) were maintained with the addition of a cryoprotectant, trehalose. The release profile of BMP-2 from FD-PEC was similar to that of freshly prepared PEC. In vitro bioactivity analysis of the released BMP-2 showed that the carrier performance of PEC was not compromised by freeze-drying up to three-month storage at room temperature. BMP-2-bound FD-PEC induced comparable bone formation to that using freshly prepared regular PEC in a rat posterolateral spinal fusion model. These results suggest that FD-PEC is capable of delivering low-dose BMP-2 and could be developed as an off-the-shelf product for translational applications. The simplicity of this preservation method provides promise for the translational application of PEC.

Synergistic Effect of NELL-1 and an Ultra-Low Dose of BMP-2 on Spinal Fusion.

Bone morphogenetic protein 2 (BMP-2) is widely used in spinal fusion but it can cause adverse effects such as ectopic bone and adipose tissue in vivo. Neural epidermal growth factor like-like molecule-1 (NELL-1) has been shown to suppress BMP-2-induced adverse effects. However, no optimum carriers that control both NELL-1 and BMP-2 releases to elicit long-term bioactivity have been developed. In this study, we employed polyelectrolyte complex (PEC) as a control release carrier for NELL-1 and BMP-2. An ultra-low dose of BMP-2 synergistically functioned with NELL-1 on bone marrow mesenchymal stem cells osteogenic differentiation with greater mineralization in vitro. The osteoinductive ability of NELL-1 and an ultra-low dose of BMP-2 in PEC was investigated in rat posterolateral spinal fusion. Our results showed increased fusion rate, bone architecture, and improved bone stiffness at 8 weeks after surgery in the combination groups compared with NELL-1 or BMP-2 alone. Moreover, the formation of ectopic bone and adipose tissue was negligible in all the PEC groups. In summary, dual delivery of NELL-1 and an ultra-low dose of BMP-2 in the PEC control release carrier has greater fusion efficiency compared with BMP-2 alone and could potentially be a better alternative to the currently used BMP-2 treatments for spinal fusion. Impact Statement In this study, polyelectrolyte complex was used to absorb neural epidermal growth factor like-like molecule-1 (NELL-1) and bone morphogenetic protein 2 (BMP-2) to achieve controlled dual release. The addition of NELL-1 significantly reduced the effective dose of BMP-2 to 2.5% of its conventional dose in absorbable collagen sponge, to produce solid spinal fusion without significant adverse effects. This study was the first to identify the efficacy of combination NELL-1 and BMP-2 in a control release carrier in spinal fusion, which could be potentially used clinically to increase fusion rate and avoid the adverse effects commonly associated with conventional BMP-2.

Bone Regeneration by Controlled Release of Bone Morphogenetic Protein-2: A Rabbit Spinal Fusion Chamber Molecular Study.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) has been widely used in spine fusion surgery. However, high doses of rhBMP-2 delivered with absorbable collagen sponge (ACS) have led to inflammation-related adverse conditions. Polyelectrolyte complex (PEC) control release carrier can substantially reduce the rhBMP-2 dose and complication without compromising fusion. The molecular events underlying controlled release and their effects on spinal fusion remain unknown. In this study, a rabbit interbody spinal fusion chamber was designed to provide a controlled environment for profiling molecular events during the fusion process. Study groups included Group 1, PEC with 100 µg rhBMP-2; Group 2, ACS with 100 µg rhBMP-2; Group 3, ACS with 300 µg rhBMP-2; Group 4, autologous bone graft; and Group 5, empty chamber. Manual palpation, microcomputed tomography, and histological analysis showed that Group 1 and 3 achieved bone fusion, while the other groups showed no signs of fusion. Gene expression profiling showed robust induction of osteogenic markers in Groups 1 and 3, with modulated early induction of inflammatory genes in the PEC group. Delivery of 100 µg rhBMP-2 with ACS (Group 2) resulted in less upregulation of osteogenic genes, increased inflammatory genes expression, and upregulation of osteoclastic genes compared to Group 1. These results suggest that the manner of BMP-2 release at the interbody spinal defect site could dictate the balance of in-situ osteogenic and antiosteogenic activities, affecting fusion outcomes. The molecular evidence supports PEC for sustained release of BMP-2 for spinal interbody fusion, and the feasibility of employing this novel interbody spinal fusion chamber for future molecular studies. Impact Statement A radiolucent rabbit interbody spinal fusion chamber was developed to study the molecular events during spinal fusion process. The gene expression profile suggests that control release of bone morphogenetic protein-2 (BMP-2) resulted in lower inflammatory and osteoclastic activities, but elicited higher osteogenic activities, while burst release of BMP-2 resulted in predominantly inflammation and osteoclastogenesis with minimum osteogenic activity. This study provides the molecular evidence that underscores the regeneration outcomes from the two different BMP-2 delivery systems. This spinal fusion chamber could be used for future molecular studies to optimize carrier design for spinal fusion.