Tough Photo-Cross-Linked PCL-Hydroxyapatite Composites for Bone Tissue Engineering.

Acrylate-based photo-cross-linked poly(ε-caprolactone) (PCL) tends to show low elongation and strength. Incorporation of osteo-inductive hydroxyapatite (HAp) further enhances this effect, which limits its applicability in bone tissue engineering. To overcome this, the thiol-ene click reaction is introduced for the first time in order to photo-cross-link PCL composites with 0, 10, 20, and 30 wt % HAp nanoparticles. It is demonstrated that the elongation at break and ultimate strength increase 10- and 2-fold, respectively, when the photopolymerization mechanism is shifted from a radical chain-growth (i.e., acrylate cross-linking) toward a radical step-growth polymerization (i.e., thiol-ene cross-linking). Additionally, it is illustrated that osteoblasts can attach to and proliferate on the surface of the photo-cross-linked PCL-HAp composites. Finally, the incorporation of HAp nanoparticles is shown to reduce the ALP activity of osteoblasts. Overall, thiol-ene cross-linked PCL-HAp composites can be considered as promising potential materials for bone tissue engineering.

Direct electrospinning of poly(vinyl butyral) onto human dermal fibroblasts using a portable device.

OBJECTIVE: To demonstrate that uniform poly(vinyl butyral) (PVB) fibres can be safely electrospun onto a monolayer of human dermal fibroblasts using a portable device. RESULTS: PVB in solvent mixtures containing various amounts of ethanol and water was electrospun. Six percent (weight-to-volume ratio) PVB in a 9:1 ethanol:water ratio was the solution with the highest content in water that could be electrospun into consistent fibres with an average diameter of 0.9 µm (± 0.1 µm). Four and five percent PVB solutions created beaded fibres. A 8:2 ethanol:water solution lead to microbead formation while a 7:3 ethanol:water mix failed to fully dissolve. The selected solution was successfully electrospun onto a monolayer of human dermal fibroblasts and the process had no significant effect (p < 0.05) on cell viability compared to the control without fibres. CONCLUSIONS: PVB-ethanol-water solutions could be electrospun without damaging the exposed cell layer. However, further work is required to demonstrate the long-term effect of PVB as a wound healing material.

Translating Regenerative Biomaterials Into Clinical Practice.

Globally health care spending is increasing unsustainably. This is especially true of the treatment of musculoskeletal (MSK) disease where in the United States the MSK disease burden has doubled over the last 15 years. With an aging and increasingly obese population, the surge in MSK related spending is only set to worsen. Despite increased funding, research and attention to this pressing health need, little progress has been made toward novel therapies. Tissue engineering and regenerative medicine (TERM) strategies could provide the solutions required to mitigate this mounting burden. Biomaterial-based treatments in particular present a promising field of potentially cost-effective therapies. However, the translation of a scientific development to a successful treatment is fraught with difficulties. These barriers have so far limited translation of TERM science into clinical treatments. It is crucial for primary researchers to be aware of the barriers currently restricting the progression of science to treatments. Researchers need to act prospectively to ensure the clinical, financial, and regulatory hurdles which seem so far removed from laboratory science do not stall or prevent the subsequent translation of their idea into a treatment. The aim of this review is to explore the development and translation of new treatments. Increasing the understanding of these complexities and barriers among primary researchers could enhance the efficiency of biomaterial translation.

Performances of a portable electrospinning apparatus.

To demonstrate that portable electrospinning devices can spin a wide range of polymers into submicron fibres and provide a mesh quality comparable to those produced with benchtop machines. We have designed a small, battery-operated electrospinning apparatus which enables control over the voltage and the flow rate of the polymer solution via a microcontroller. It can be used to electrospin a range of commonly used polymers including poly(ε-caprolactone), poly(p-dioxanone), poly(lactic-co-glycolic acid), poly(3-hydroxybutyrate), poly(ethylene oxide), poly(vinyl acohol) and poly(vinyl butyral). Moreover, electrospun meshes are produced with a quality comparable to a benchtop machine. We also show that the portable apparatus is able to electrospray beads and microparticles. Finally, we highlight the potential of the device for wound healing applications by demonstrating the possibility of electrospinning onto pig and human skins. Portable electrospinning devices are still at an early stage of development but they could soon become an attractive alternative to benchtop machines, in particular for uses that require mobility and a higher degree of flexibility, such as for wound healing applications.

Soft bioreactor systems: a necessary step toward engineered MSK soft tissue?

A key objective of tissue engineering (TE) is to produce in vitro funcional grafts that can replace damaged tissues or organs in patients. TE uses bioreactors, which are controlled environments, allowing the application of physical and biochemical cues to relevant cells growing in biomaterials. For soft musculoskeletal (MSK) tissues such as tendons, ligaments and cartilage, it is now well established that applied mechanical stresses can be incorporated into those bioreactor systems to support tissue growth and maturation via activation of mechanotransduction pathways. However, mechanical stresses applied in the laboratory are often oversimplified compared to those found physiologically and may be a factor in the slow progression of engineered MSK grafts towards the clinic. In recent years, an increasing number of studies have focused on the application of complex loading conditions, applying stresses of different types and direction on tissue constructs, in order to better mimic the cellular environment experienced in vivo. Such studies have highlighted the need to improve upon traditional rigid bioreactors, which are often limited to uniaxial loading, to apply physiologically relevant multiaxial stresses and elucidate their influence on tissue maturation. To address this need, soft bioreactors have emerged. They employ one or more soft components, such as flexible soft chambers that can twist and bend with actuation, soft compliant actuators that can bend with the construct, and soft sensors which record measurements in situ. This review examines types of traditional rigid bioreactors and their shortcomings, and highlights recent advances of soft bioreactors in MSK TE. Challenges and future applications of such systems are discussed, drawing attention to the exciting prospect of these platforms and their ability to aid development of functional soft tissue engineered grafts.

A Hybrid Electrospun-Extruded Polydioxanone Suture for Tendon Tissue Regeneration.

Many surgical tendon repairs fail despite advances in surgical materials and techniques. Tendon repair failure can be partially attributed to the tendon's poor intrinsic healing capacity and the repurposing of sutures from other clinical applications. Electrospun materials show promise as a biological scaffold to support endogenous tendon repair, but their relatively low tensile strength has limited their clinical translation. It is hypothesized that combining electrospun fibers with a material with increased tensile strength may improve the suture's mechanical properties while retaining biophysical cues necessary to encourage cell-mediated repair. This article describes the production of a hybrid electrospun-extruded suture with a sheath of submicron electrospun fibers and a core of melt-extruded fibers. The porosity and tensile strength of this hybrid suture is compared with an electrospun-only braided suture and clinically used sutures Vicryl and polydioxanone (PDS). Bioactivity is assessed by measuring the adsorbed serum proteins on electrospun and melt-extruded filaments using mass spectrometry. Human hamstring tendon fibroblast attachment and proliferation were quantified and compared between the hybrid and control sutures. Combining an electrospun sheath with melt-extruded cores created a hybrid braid with increased tensile strength (70.1 ± 0.3N) compared with an electrospun only suture (12.9 ± 1 N, p < 0.0001). The hybrid suture had a similar force at break to clinical sutures, but lower stiffness and stress. The Young's modulus was 772.6 ± 32 MPa for the hybrid suture, 1693.0 ± 69 MPa for PDS, and 3838.0 ± 132 MPa for Vicryl, p < 0.0001. Hybrid sutures had lower overall porosity than electrospun-only sutures (40 ± 4% and 60 ± 7%, respectively, p = 0.0018) but had a significantly larger overall porosity and average pore diameter compared with surgical sutures. There were similar clusters of adsorbed proteins on electrospun and melt-extruded filaments, which were distinct from PDS. Tendon fibroblast attachment and cell proliferation on hybrid and electrospun sutures were significantly higher than on clinical sutures. This study demonstrated that a bioactive suture with increased tensile strength and lower stiffness could be produced by adding a core of 10 µm melt-extruded fibers to a sheath of electrospun fibers. In contrast to currently used sutures, the hybrid sutures promoted a bioactive response: serum proteins adsorbed, and fibroblasts attached, survived, grew along the sutures, and adopted appropriate morphologies.

Synthetic and degradable patches: an emerging solution for rotator cuff repair.

The use of rotator cuff augmentation has increased dramatically over the last 10 years in response to the high rate of failure observed after non-augmented surgery. However, although augmentations have been shown to reduce shoulder pain, there is no consensus or clear guideline as to what is the safest or most efficacious material. Current augmentations, either available commercially or in development, can be classified into three categories: non-degradable structures, extra cellular matrix (ECM)-based patches and degradable synthetic scaffolds. Non-degradable structures have excellent mechanical properties, but can cause problems of infection and loss of integrity in the long-term. ECM-based patches usually demonstrate excellent biological properties in vitro, but studies have highlighted complications in vivo due to poor mechanical support and to infection or inflammation. Degradable synthetic scaffolds represent the new generation of implants. It is proposed that a combination of good mechanical properties, active promotion of biological healing, low infection risk and bio-absorption are the ideal characteristics of an augmentation material. Among the materials with these features, those processed by electrospinning have shown great promis. However, their clinical effectiveness has yet to be proven and well conducted clinical trials are urgently required.

Time-dependent extracellular matrix alterations of young tendons in response to stress relaxation: a model for the Ponseti method.

The Ponseti method corrects a clubfoot by manipulation and casting which causes stress relaxation on the tendons. Here, we examined the effect of long-term stress relaxation on tendon extracellular matrix (ECM) by (1) an ex vivo stress relaxation test, (2) an in vitro tenocyte culture with stress relaxation and (3) an in vivo rabbit study. Time-dependent tendon lengthening and ECM alterations including crimp angle reduction and cleaved elastin were observed, which illustrated the mechanism of tissue lengthening behind the treatment-a material-based crimp angle reduction resulted from elastin cleavage. Additionally, in vitro and in vivo results observed restoration of these ECM alterations along with increased elastin level after 7 days of treatment, and the existence of neovascularization and inflammation, indicating the recovery and adaptation from the tendon in reaction to the treatment. Overall, this study provides the scientific background and information that helps explain the Ponseti method.

Manufacture of Soft-Hard Implants from Electrospun Filaments Embedded in 3D Printed Structures.

Rotator cuff tendon tears are common injuries of the musculoskeletal system that often require surgical repair. However, re-tearing following repair is a significant clinical problem, with a failure rate of up to 40%, notably at the transition from bone to tendon. The development of biphasic materials consisting of soft and hard components, which can mimic this interface, is therefore promising. Here, a simple manufacturing approach is proposed that combines electrospun filaments and 3D printing to achieve scaffolds made of a soft polydioxanone cuff embedded in a porous polycaprolactone block. The insertion area of the cuff is based on the supraspinatus tendon footprint and the size of the cuff is scaled up from 9 to 270 electrospun filaments to reach a clinically relevant strength of 227N on average. The biological evaluation shows that the biphasic scaffold components are noncytotoxic, and that tendon and bone cells can be grown on the cuff and block, respectively. Overall, these results indicate that combining electrospinning and 3D printing is a feasible and promising approach to create soft-to-hard biphasic scaffolds that can improve the outcomes of rotator cuff repair.

Advanced Robotics to Address the Translational Gap in Tendon Engineering.

Tendon disease is a significant and growing burden to healthcare systems. One strategy to address this challenge is tissue engineering. A widely held view in this field is that mechanical stimulation provided to constructs should replicate the mechanical environment of native tissue as closely as possible. We review recent tendon tissue engineering studies in this article and highlight limitations of conventional uniaxial tensile bioreactors used in current literature. Advanced robotic platforms such as musculoskeletal humanoid robots and soft robotic actuators are promising technologies which may help address translational gaps in tendon tissue engineering. We suggest the proposed benefits of these technologies and identify recent studies which have worked to implement these technologies in tissue engineering. Lastly, key challenges to address in adapting these robotic technologies and proposed future research directions for tendon tissue engineering are discussed.

In vitro evaluation of the response of human tendon-derived stromal cells to a novel electrospun suture for tendon repair.

Recurrent tears after surgical tendon repair remain common. Repair failures can be partly attributed to the use of sutures not designed for the tendon cellular niche nor for the promotion of repair processes. Synthetic electrospun materials can mechanically support the tendon whilst providing topographical cues that regulate cell behaviour. Here, a novel electrospun suture made from twisted polydioxanone (PDO) polymer filaments is compared to PDS II, a clinically-used PDO suture currently utilised in tendon repair. We evaluated the ability of these sutures to support the attachment and proliferation of human tendon-derived stromal cells using PrestoBlue and Scanning Electron Microscopy. Suture surface chemistry was analysed using X-ray Photoelectron Spectroscopy. Bulk RNA-Seq interrogated the transcriptional response of primary tendon-derived stromal cells to sutures after 14 days. Electrospun suture showed increased initial cell attachment and a stronger transcriptional response compared to PDS II, with relative enrichment of pathways including mTorc1 signalling and depletion of epithelial mesenchymal transition. Neither suture induced transcriptional upregulation of inflammatory pathways compared to baseline. Twisted electrospun sutures therefore show promise in improving outcomes in surgical tendon repair by allowing increased cell attachment whilst maintaining an appropriate tissue response.

Early development of a polycaprolactone electrospun augment for anterior cruciate ligament reconstruction.

Despite the clinical success of Anterior Cruciate Ligament reconstruction (ACLR) in some patients, unsatisfactory clinical outcomes secondary to graft failure are seen, indicating the need to develop new regeneration strategies. The use of degradable and bioactive textiles has the potential to improve the biological repair of soft tissue. Electrospun (ES) filaments are particularly promising as they have the ability to mimic the structure of natural tissues and influence endogenous cell behaviour. In this study, we produced continuous polycaprolactone (PCL) ES filaments using a previously described electrospinning collection method. These filaments were stretched, twisted, and assembled into woven structures. The morphological, tensile, and biological properties of the woven fabric were then assessed. Scanning electron microscopy (SEM) images highlighted the aligned and ACL-like microfibre structure found in the stretched filaments. The tensile properties indicated that the ES fabric reached suitable strengths for a use as an ACLR augmentation device. Human ACL-derived cell cultured on the fabric showed approximately a 3-fold increase in cell number over 2 weeks and this was equivalent to a collagen coated synthetic suture commonly used in ACLR. Cells generally adopted a more elongated cell morphology on the ES fabric compared to the control suture, aligning themselves in the direction of the microfibres. A NRU assay confirmed that the ES fabric was non-cytotoxic according to regulatory standards. Overall, this study supports the development of ES textiles as augmentation devices for ACLR.

Histological evaluation of cellular response to a multifilament electrospun suture for tendon repair.

BACKGROUND: Rotator cuff tendon repair in humans is a commonly performed procedure aimed at restoring the tendon-bone interface. Despite significant innovation of surgical techniques and suture anchor implants, only 60% of repairs heal successfully. One strategy to enhance repair is the use of bioactive sutures that provide the native tendon with biophysical cues for healing. We investigated the tissue response to a multifilament electrospun polydioxanone (PDO) suture in a sheep tendon injury model characterised by a natural history of failure of healing. METHODOLOGY AND RESULTS: Eight skeletally mature English Mule sheep underwent repair with electrospun sutures. Monofilament sutures were used as a control. Three months after surgery, all tendon repairs healed, without systemic features of inflammation, signs of tumour or infection at necropsy. A mild local inflammatory reaction was seen. On histology the electrospun sutures were densely infiltrated with predominantly tendon fibroblast-like cells. In comparison, no cellular infiltration was observed in the control suture. Neovascularisation was observed within the electrospun suture, whilst none was seen in the control. Foreign body giant cells were rarely seen with either sutures. CONCLUSION: This study demonstrates that a tissue response can be induced in tendon with a multifilament electrospun suture with no safety concerns.

Histopathological and immunohistochemical evaluation of cellular response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch for tendon repair.

We investigated endogenous tissue response to a woven and electrospun polydioxanone (PDO) and polycaprolactone (PCL) patch intended for tendon repair. A sheep tendon injury model characterised by a natural history of consistent failure of healing was chosen to assess the biological potential of woven and aligned electrospun fibres to induce a reparative response. Patches were implanted into 8 female adult English Mule sheep. Significant infiltration of tendon fibroblasts was observed within the electrospun component of the patch but not within the woven component. The cellular infiltrate into the electrospun fibres was accompanied by an extensive network of new blood vessel formation. Tendon fibroblasts were the most abundant scaffold-populating cell type. CD45+, CD4+ and CD14+ cells were also present, with few foreign body giant cells. There were no local or systemic signs of excessive inflammation with normal hematology and serology for inflammatory markers three months after scaffold implantation. In conclusion, we demonstrate that an endogenous healing response can be safely induced in tendon by means of biophysical cues using a woven and electrospun patch.

Short Overview of ROS as Cell Function Regulators and Their Implications in Therapy Concepts.

The importance of reactive oxygen species (ROS) has been gradually acknowledged over the last four decades. Initially perceived as unwanted products of detrimental oxidative stress, they have been upgraded since, and now ROS are also known to be essential for the regulation of physiological cellular functions through redox signaling. In the majority of cases, metabolic demands, along with other stimuli, are vital for ROS formation and their actions. In this review, we focus on the role of ROS in regulating cell functioning and communication among themselves. The relevance of ROS in therapy concepts is also addressed here.

Pyridine as an additive to improve the deposition of continuous electrospun filaments.

Electrospun filaments are leading to a new generation of medical yarns that have the ability to enhance tissue healing through their biophysical cues. We have recently developed a technology to fabricate continuous electrospun filaments by depositing the submicron fibres onto a thin wire. Here we investigate the influence of pyridine on the fibre deposition. We have added pyridine to polydioxanone solutions at concentrations ranging from 0 to 100 ppm, increasing the conductivity of the solutions almost linearly from 0.04 uS/cm to 7 uS/cm. Following electrospinning, this led to deposition length increasing from 1 cm to 14 cm. The samples containing pyridine easily underwent cold drawing. The strength of drawn filaments increased from 0.8 N to 1.5 N and this corresponded to a decrease in fibre diameter, with values dropping from 2.7 µm to 1 µm. Overall, these findings are useful to increase the reliability of the manufacturing process of continuous electrospun filaments and to vary their biophysical properties required for their application as medical yarns such as surgical sutures.

Growing tissue grafts on humanoid robots: A future strategy in regenerative medicine?

Humanoid robots may enhance growth of musculoskeletal tissue grafts for tissue transplant applications.

Effect of annealing on the mechanical properties and the degradation of electrospun polydioxanone filaments.

Annealing, or heat treatment, has traditionally been used as a treatment to improve the strength and stiffness of electrospun materials. Understanding the extent to which annealing can improve the mechanical properties and alter the degradation rate of electrospun polydioxanone filaments could influence the range of its potential clinical applications. In this study, we investigated the effect of annealing electrospun polydioxanone filaments at varying times and temperatures and subsequently subjecting them to in vitro degradation in phosphate buffer saline for up to 6 weeks. Fibre alignment, tensile strength and thermal properties were assessed. It was determined that annealing at 65°C for 3h only marginally improved the tensile strength (9±2%) but had a significant effect on reducing strain and rate of degradation, as well as maintaining fibre alignment within the filament. The filament retained significantly more of its force at failure after 4 weeks (82±15%, compared to 61±20% for non annealed filaments) and after 6 weeks of degradation (81±9%, compared to 55±13% for non annealed filaments). Conversely, annealing filaments at 75°C improved the initial tensile strength of the filament (17±6%), but over 6 weeks, both samples annealed at 75°C and 85°C otherwise performed similarly or mechanically worse than those not annealed. These findings suggest that annealing at low temperatures is more useful as a method to tailor degradation rate than to improve mechanical properties. The ability to modulate the degradation profile with annealing may become useful to tailor the properties of electrospun materials without altering the chemistry of the polymer used. This might better match the degradation of the implant and gradual loss of mechanical properties with the new matrix deposition within the structure, enabling multiple regenerative strategies within a single biomaterial system.

Investigating the use of curcumin-loaded electrospun filaments for soft tissue repair applications.

Electrospun filaments represent a new generation of medical textiles with promising applications in soft tissue repair. A potential strategy to improve their design is to combine them with bioactive molecules. Curcumin, a natural compound found in turmeric, is particularly attractive for its antioxidant, anti-inflammatory, and antimicrobial properties. However, investigating the range of relevant doses of curcumin in materials designed for tissue regeneration has remained limited. In this paper, a wide range of curcumin concentrations was explored and the potential of the resulting materials for soft tissue repair applications was assessed. Polydioxanone (PDO) filaments were prepared with various amounts of curcumin: 0%, 0.001%, 0.01%, 0.1%, 1%, and 10% (weight to weight ratio). The results from the present study showed that, at low doses (≤0.1%), the addition of curcumin has no influence on the spinning process or on the physicochemical properties of the filaments, whereas higher doses lead to smaller fiber diameters and improved mechanical properties. Moreover, filaments with 0.001% and 0.01% curcumin stimulate the metabolic activity and proliferation of normal human dermal fibroblasts (NHDFs) compared with the no-filament control. However, this stimulation is not significant when compared to the control filaments (0%). Highly dosed filaments induce either the inhibition of proliferation (with 1%) or cell apoptosis (with 10%) as a result of the concentrations of curcumin found in the medium (9 and 32 µM, respectively), which are near or above the known toxicity threshold of curcumin (~10 µM). Moreover, filaments with 10% curcumin increase the catalase activity and glutathione content in NHDFs, indicating an increased production of reactive oxygen species resulting from the large concentration of curcumin. Overall, this study suggested that PDO electrospun filaments loaded with low amounts of curcumin are more promising compared with higher concentrations for stimulating tissue repair. This study also highlighted the need to explore lower concentrations when using polymers as PDO, such as those with polycaprolactone and other degradable polyesters.

Synthetic sutures: Clinical evaluation and future developments.

Today's sutures are the result of a 4000-year innovation process with regard to their materials and manufacturing techniques, yet little has been done to enhance the therapeutic value of the suture itself. In this review, we explore the historical development, regulatory database and clinical literature of sutures to gain a fuller picture of suture advances to date. First, we examine historical shifts in suture manufacturing companies and review suture regulatory databases to understand the forces driving suture development. Second, we gather the existing clinical evidence of suture efficacy from reviewing the clinical literature and the Food and Drug Administration database in order to identify to what extent sutures have been clinically evaluated and the key clinical areas that would benefit from improved suture materials. Finally, we apply tissue engineering and regenerative medicine design hypotheses to suture materials to identify routes by which bioactive sutures can be designed and passed through regulatory hurdles, to improve surgical outcomes. Our review of the clinical literature revealed that many of the sutures currently in use have been available for decades, yet have never been clinically evaluated. Since suture design and development is industry driven, incremental modifications have allowed for a steady outflow of products while maintaining a safe regulatory position and limiting costs. Until recently, there has been little academic interest in suture development, however the rise of regenerative medicine strategies is shifting the suture paradigm from an inert material, which mechanically approximates tissue, to a bioactive material, which also actively promotes cell-directed repair and a positive healing response. These materials hold significant therapeutic potential, but could be associated with an increased regulatory burden, cost, and clinical evaluation compared with current devices.