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.

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.

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.

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.

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.

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.

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.

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.

Antagonist muscle co-contraction during a double-leg landing maneuver at two heights.

BACKGROUND: Knee injuries are common during landing activities. Greater landing height increases peak ground reaction forces (GRFs) and loading at the knee joint. As major muscles to stabilize the knee joint, Quadriceps and Hamstring muscles provide internal forces to attenuate the excessive GRF. Despite the number of investigations on the importance of muscle function during landing, the role of landing height on these muscles forces using modeling during landing is not fully investigated. METHODS: Participant-specific musculoskeletal models were developed using experimental motion analysis data consisting of anatomic joint motions and GRF from eight male participants performing double-leg drop landing from 30 and 60 cm. Muscle forces were calculated in OpenSim and their differences were analyzed at the instances of high risk during landing i.e. peak GRF for both heights. RESULTS: The maximum knee flexion angle and moments were found significantly higher from a double-leg landing at 60 cm compared to 30 cm. The results showed elevated GRF, and mean muscle forces during landing. At peak GRF, only quadriceps showed significantly greater forces at 60 cm. Hamstring muscle forces did not significantly change at 60 cm compared to 30 cm. CONCLUSIONS: Quadriceps and hamstring muscle forces changed at different heights. Since hamstring forces were similar in both landing heights, this could lead to an imbalance between the antagonist muscles, potentially placing the knee at risk of injury if combined with small flexion angles that was not observed at peak GRF in our study. Thus, enhanced neuromuscular training programs strengthening the hamstrings may be required to address this imbalance. These findings may contribute to enhance neuromuscular training programs to prevent knee injuries during landing.

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.

A Magnetic Resonance Compatible Soft Wearable Robotic Glove for Hand Rehabilitation and Brain Imaging.

In this paper, we present the design, fabrication and evaluation of a soft wearable robotic glove, which can be used with functional Magnetic Resonance imaging (fMRI) during the hand rehabilitation and task specific training. The soft wearable robotic glove, called MR-Glove, consists of two major components: a) a set of soft pneumatic actuators and b) a glove. The soft pneumatic actuators, which are made of silicone elastomers, generate bending motion and actuate finger joints upon pressurization. The device is MR-compatible as it contains no ferromagnetic materials and operates pneumatically. Our results show that the device did not cause artifacts to fMRI images during hand rehabilitation and task-specific exercises. This study demonstrated the possibility of using fMRI and MR-compatible soft wearable robotic device to study brain activities and motor performances during hand rehabilitation, and to unravel the functional effects of rehabilitation robotics on brain stimulation.

Anisotropic silk fibroin/gelatin scaffolds from unidirectional freezing.

Recent studies have underlined the importance of matching scaffold properties to the biological milieu. Tissue, and thus scaffold, anisotropy is one such property that is important yet sometimes overlooked. Methods that have been used to achieve anisotropic scaffolds present challenges such as complicated fabrication steps, harsh processing conditions and toxic chemicals involved. In this study, unidirectional freezing was employed to fabricate anisotropic silk fibroin/gelatin scaffolds in a simple and mild manner. Morphological, mechanical, chemical and cellular compatibility properties were investigated, as well as the effect of the addition of gelatin to certain properties of the scaffold. It was shown that scaffold properties were suitable for cell proliferation and that mesenchymal stem cells were able to align themselves along the directed fibers. The fabricated scaffolds present a platform that can be used for anisotropic tissue engineering applications such as cardiac patches.

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.

Temporal profiling of the growth and multi-lineage potentiality of adipose tissue-derived mesenchymal stem cells cell-sheets.

Cell-sheet tissue engineering retains the benefits of an intact extracellular matrix (ECM) and can be used to produce scaffold-free constructs. Adipose tissue-derived stem cells (ASCs) are multipotent and more easily obtainable than the commonly used bone marrow-derived stem cells (BMSCs). Although BMSC cell sheets have been previously reported to display multipotentiality, a detailed study of the development and multilineage potential of ASC cell sheets (ASC-CSs) is non-existent in the literature. The aims of this study were to temporally profile: (a) the effect of hyperconfluent culture duration on ASC-CSs development; and (b) the multipotentiality of ASC-CSs by differentiation into the osteogenic, adipogenic and chondrogenic lineages. Rabbit ASCs were first isolated and cultured until confluence (day 0). The confluent cells were then cultured in ascorbic acid-supplemented medium for 3 weeks to study cell metabolic activity, cell sheet thickness and early differentiation gene expressions at weekly time points. ASC-CSs and ASCs were then differentiated into the three lineages, using established protocols, and assessed by RT-PCR and histology at multiple time points. ASC-CSs remained healthy up to 3 weeks of hyperconfluent culture. One week-old cell sheets displayed upregulation of early differentiation gene markers (Runx2 and Sox9); however, subsequent differentiation results indicated that they did not necessarily translate to an improved phenotype. ASCs within the preformed cell sheet groups did not differentiate as efficiently as the non-hyperconfluent ASCs, which were directly differentiated. Although ASCs within the cell sheets retained their differentiation capacity and remained viable under prolonged hyperconfluent conditions, future applications of ASC-CSs in tissue engineering should be considered with care. Copyright © 2016 John Wiley & Sons, Ltd.

Bone Marrow Derived Mesenchymal Stem Cell Augmentation of Rabbit Flexor Tendon Healing.

BACKGROUND: This study investigated the effect of mesenchymal stem cell implantation on flexor tendon healing using a rabbit model of flexor tendon repair. Specifically, we compared the difference between autologous and allogeneic stem cells. The influence of cell number on the outcome of flexor tendon healing was also investigated. METHODS: Repaired tendons on the rear paws of rabbits were randomly assigned into four groups: control group, 1 million autologous cells, 1 million allogeneic cells, and 4 million allogeneic cells. Rabbits were sacrificed at 3 or 8 weeks after surgery. RESULTS: Implantation of 4 million stem cells resulted in a significant increase in range of motion compared with control group at three weeks after surgery. The positive staining of collagen I in healing tendons was enhanced in stem cell treated groups three weeks after surgery. However, stem cells did not improve biomechanical properties of flexor tendons. CONCLUSIONS: High dose stem cells attenuated adhesions in the early time point following flexor tendon repair. Further work is needed determine the value of stem cell therapy in flexor tendon healing in humans.

Bone marrow-derived mesenchymal stem cells assembled with low-dose BMP-2 in a three-dimensional hybrid construct enhances posterolateral spinal fusion in syngeneic rats.

BACKGROUND CONTEXT: The combination of potent osteoinductive growth factor, functional osteoblastic cells, and osteoconductive materials to induce bone formation is a well-established concept in bone tissue engineering. However, supraphysiological dose of growth factor, such as recombinant human bone morphogenetic protein 2 (rhBMP-2), which is necessary in contemporary clinical application, have been reported to result in severe side effects. PURPOSE: We hypothesize that the synergistic osteoinductive capacity of low-dose bone morphogenetic protein 2 (BMP-2) combined with undifferentiated bone marrow-derived stromal cells (BMSCs) is comparable to that of osteogenically differentiated BMSCs when used in a rodent model of posterolateral spinal fusion. STUDY DESIGN/SETTING: A prospective study using a rodent model of posterolateral spinal fusion was carried out. PATIENT SAMPLE: Thirty-six syngeneic Fischer rats comprised the patient sample. METHODS: Six groups of implants were evaluated as follows (n=6): (1) 10 µg BMP-2 with undifferentiated BMSCs; (2) 10 µg BMP-2 with osteogenic-differentiated BMSCs; (3) 2.5 µg BMP-2 with undifferentiated BMSCs; (4) 2.5 µg BMP-2 with osteogenic-differentiated BMSCs; (5) 0.5 µg BMP-2 with undifferentiated BMSCs; and (6) 0.5 µg BMP-2 with osteogenic-differentiated BMSCs. Optimal in vitro osteogenic differentiation of BMSCs was determined by quantitative real-time polymerase chain reaction (qRT-PCR) gene analysis whereas in vivo bone formation capacity was evaluated by manual palpation, micro-computed tomography, and histology. RESULTS: Rat BMSCs cultured in fibrin matrix that was loaded into the pores of medical-grade poly epsilon caprolactone tricalcium phosphate scaffolds differentiated toward osteogenic lineage by expressing osterix, runt-related transcription factor 2, and osteocalcium mRNA when supplemented with dexamethasone, ascorbic acid, and ß-glycerophosphate. Whereas qRT-PCR revealed optimal increase in osteogenic genes expression after 7 days of in vitro culture, in vivo transplantation study showed that pre-differentiation of BMSCs before transplantation failed to promote posterolateral spinal fusion when co-delivered with low-dose BMP-2 (1/6 or 17% fusion rate). In contrast, combined delivery of undifferentiated BMSCs with low-dose BMP-2 (2.5 µg) demonstrated significantly higher fusion rate (4/6 or 67%) as well as significantly increased volume of new bone formation (p<.05). CONCLUSION: In summary, this study supports the combination of undifferentiated BMSCs and low-dose rhBMP-2 for bone tissue engineering construct.

Polyelectrolyte Complex Carrier Enhances Therapeutic Efficiency and Safety Profile of Bone Morphogenetic Protein-2 in Porcine Lumbar Interbody Fusion Model.

STUDY DESIGN: Porcine lumbar interbody fusion model. OBJECTIVE: This study evaluates the effect of polyelectrolyte complex (PEC) carrier in enhancing the therapeutic efficiency and safety profile of bone morphogenetic protein-2 (BMP-2) in a large animal model. SUMMARY OF BACKGROUND DATA: Extremely large amounts of BMP-2 are administered to achieve consistent spinal fusion, which has led to complications. Heparin-modified PEC carrying reduced BMP-2 doses of 0.5 µg was demonstrated to achieve consistent spinal fusion with reduction of complications in rodent model. The purpose of this study was to evaluate whether PEC could improve the therapeutic efficiency of BMP-2 in porcine model. METHODS: Three-segment (L3-L6) anterior lumbar interbody fusions with instrumentation were performed on 6 pigs using 3 different doses of BMP-2, namely, (1) 50 µg, (2) 150 µg, and (3) 300 µg. The BMP-2 was delivered using heparin-modified alginate microbeads loaded into biodegradable cage. Fusion performance was evaluated after 3 months. RESULTS: Manual palpation and micro-computed tomography showed consistent fusion in all experimental groups. Heterotopic bone formation beyond the cage implant area was more evident in group 2 and group 3 than in group 1. Similarly, superior bone microstructure was observed in the new bone with the lowered BMP-2 dose. Biomechanical evaluation revealed enhanced stiffness of the operated segments compared with nonoperated segments (P < 0.05). Mechanical stability was maintained despite dose reduction of BMP-2. Although the mineral apposition rate was higher in group 3, unsatisfactory bony microstructure with decreased trabecular number was observed in group 3 compared with group 1. CONCLUSION: PEC carrying low doses of BMP-2 achieved consistent interbody fusion. We observed dose-related reduction in heterotopic ossification without compromising the stability of the fused segments. PEC carrier reduces the efficacious doses of BMP-2. This could enhance the safety profile of BMP-2 and reduce dose- and carrier-related complications. LEVEL OF EVIDENCE: N/A.

Novel Protamine-Based Polyelectrolyte Carrier Enhances Low-Dose rhBMP-2 in Posterolateral Spinal Fusion.

STUDY DESIGN: A rodent posterolateral spinal fusion model. OBJECTIVE: This study evaluated a protamine-based polyelectrolyte complex (PEC) developed to use heparin in enhancing the biological activity of low-dose recombinant human bone morphogenetic protein-2 (rhBMP-2) in spinal fusion. SUMMARY OF BACKGROUND DATA: rhBMP-2 is commonly regarded as the most potent bone-inducing molecule. However, poor pharmacokinetics and short in vivo half-life means that large amounts of the bioactive growth factor are required for consistent clinical outcomes. This has been associated with a number of adverse tissue reactions including seroma and heterotopic ossification. Glycosaminoglycans including heparin are known to stabilize rhBMP-2 bioactivity. Previous studies with poly-L-lysine (PLL) and heparin-based PEC carriers amplified the therapeutic efficacy of low-dose BMP-2. However, questions remained on the eventual clinical applicability of relatively cytotoxic PLL. In the present study, a protamine-based PEC carrier was designed to further enhance the safety and efficacy of BMP-2 by delivering lower dose within the therapeutic window. METHODS: A polyelectrolyte shell was deposited on the surface of alginate microbead templates using the polycation (protamine)/polyanion (heparin) layer-by-layer polyelectrolyte self-assembly protocol. rhBMP-2 was loaded onto the outermost layer via heparin affinity binding. Loading and release of rhBMP-2 were evaluated in vitro. The bone-inductive ability of 20-fold reduction of rhBMP-2 with the different carrier vehicle was evaluated using a posterolateral spinal fusion model in rats. RESULTS: In vitro uptake and release analysis, protamine-based PEC showed higher uptake and significantly enhanced control release than PLL-based PEC (P < 0.05). In vivo implantation with protamine-based and PLL-based PEC showed better fusion performances than absorbable collagen sponge-delivered same dose of rhBMP-2, and negative control group through manual palpation, micro-computed tomography, and histological analyses. CONCLUSION: Solid posterolateral spinal fusion was achieved with 20-fold reduction of rhBMP-2 when delivered using protamine-based PEC carrier in the rat posterolateral spinal fusion model. LEVEL OF EVIDENCE: N/A.

Enhancing analysis of cells and proteins by fluorescence imaging on silk-based biomaterials: modulating the autofluorescence of silk.

Silk is a versatile and established biomaterial for various tissue engineering purposes. However, it also exhibits strong autofluorescence signals-thereby hindering fluorescence imaging analysis of cells and proteins on silk-derived biomaterials. Sudan Black B (SB) is a lysochrome dye commonly used to stain lipids in histology. It has also been reported to be able to quench autofluorescence of tissues in histology and has been tested on artificial biomedical polymers in recent years. It was hypothesized that SB would exert similar quenching effects on silk, modulating the autofluorescence signals, and thereby enabling improved imaging analysis of cells and molecules of interests. The quenching effect of SB on the intrinsic fluorescence properties of silk and on commercial fluorescent dyes were first investigated in this study. SB was then incorporated into typical fluorescence-based staining protocols to study its effectiveness in improving fluorescence-based imaging of the cells and proteins residing with the silk-based biomaterials. Silk processed into various forms of biomaterials (e.g., films, sponges, fibers, and electrospun mats) was seeded with cells and cultured in vitro. At sacrificial time points, specimens were harvested, fixed, and prepared for fluorescence staining. SB, available commercially as a powder, was dissolved in 70% ethanol (0.3% [w/v]) to form staining solutions. SB treatment was introduced at the last step of typical immunofluorescence staining protocols for 15-120 min. For actin staining protocols by phalloidin toxin, SB staining solutions were added before and after permeabilization with Triton-X for 15-30 min. Results showed that ideal SB treatment duration is about 15 min. Apart from being able to suppress the autofluorescence of silk, this treatment duration was also not too long to adversely affect the fluorescent labeling probes used. The relative improvement brought about by SB treatment was most evident in the blue and green emission wavelengths compared with the red emission wavelength. This study has showed that the use of SB is a cost and time effective approach to enhance fluorescence-based imaging analyses of cell-seeded silk biomaterials, which otherwise would have been hindered by the unmodulated autofluorescence signals.