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Hierarchical collagen fibers are the primary source of strength in tendons and ligaments; however, these fibers largely do not regenerate after injury or with repair, resulting in limited treatment options. We previously developed a static culture system that guides ACL fibroblasts to produce native-sized fibers and early fascicles by 6 weeks. These constructs are promising ligament replacements, but further maturation is needed. Mechanical cues are critical for development in vivo and in engineered tissues; however, the effect on larger fiber and fascicle formation is largely unknown. Our objective was to investigate whether intermittent cyclic stretch, mimicking rapid muscle activity, drives further maturation in our system to create stronger engineered replacements and to explore whether cyclic loading has differential effects on cells at different degrees of collagen organization to better inform engineered tissue maturation protocols. Constructs were loaded with an established intermittent cyclic loading regime at 5 or 10 % strain for up to 6 weeks and compared to static controls. Cyclic loading drove cells to increase hierarchical collagen organization, collagen crimp, and tissue tensile properties, ultimately producing constructs that matched or exceeded immature ACL properties. Further, the effect of loading on cells varied depending on degree of organization. Specifically, 10 % load drove early improvements in tensile properties and composition, while 5 % load was more beneficial later in culture, suggesting a shift in mechanotransduction. This study provides new insight into how cyclic loading affects cell-driven hierarchical fiber formation and maturation, which will help to develop better rehabilitation protocols and engineer stronger replacements. Collagen fibers are the primary source of strength and function in tendons and ligaments throughout the body. These fibers have limited regenerate after injury, with repair, and in engineered replacements, reducing treatment options. Cyclic load has been shown to improve fibril level alignment, but its effect at the larger fiber and fascicle length-scale is largely unknown. Here, we demonstrate intermittent cyclic loading increases cell-driven hierarchical fiber formation and tissue mechanics, producing engineered replacements with similar organization and mechanics as immature ACLs. This study provides new insight into how cyclic loading affects cell-driven fiber maturation. A better understanding of how mechanical cues regulate fiber formation will help to develop better engineered replacements and rehabilitation protocols to drive repair after injury. [Display omitted] [ABSTRACT FROM AUTHOR]

Entheses are complex attachments that translate load between elastic-ligaments and stiff-bone via organizational and compositional gradients. Neither natural healing, repair, nor engineered replacements restore these gradients, contributing to high re-tear rates. Previously, we developed a culture system which guides ligament fibroblasts in high-density collagen gels to develop early postnatal-like entheses, however further maturation is needed. Mechanical cues, including slow growth elongation and cyclic muscle activity, are critical to enthesis development in vivo but these cues have not been widely explored in engineered entheses and their individual contribution to maturation is largely unknown. Our objective here was to investigate how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, individually drive enthesis maturation in our system so to shed light on the cues governing enthesis development, while further developing our tissue engineered replacements. Interestingly, we found these loads differentially drive organizational maturation, with slow stretch driving improvements in the interface/enthesis region, and cyclic load improving the ligament region. However, despite differentially affecting organization, both loads produced improvements to interface mechanics and zonal composition. This study provides insight into how mechanical cues differentially affect enthesis development, while producing some of the most organized engineered enthesis to date. Entheses attach ligaments to bone and are critical to load transfer; however, entheses do not regenerate with repair or replacement, contributing to high re-tear rates. Mechanical cues are critical to enthesis development in vivo but their individual contribution to maturation is largely unknown and they have not been widely explored in engineered replacements. Here, using a novel culture system, we provide new insight into how slow stretch, mimicking ACL growth rates, and intermittent cyclic loading, mimicking muscle activity, differentially affect enthesis maturation in engineered ligament-to-bone tissues, ultimately producing some of the most organized entheses to date. This system is a promising platform to explore cues regulating enthesis formation so to produce functional engineered replacements and better drive regeneration following repair. [Display omitted] [ABSTRACT FROM AUTHOR]

The primary source of strength in menisci, tendons, and ligaments are hierarchical collagen fibers; however, these fibers are not regenerated after injury nor in engineered replacements, resulting in limited repair options. Collagen strength is reliant on fiber alignment, density, diameter, and crosslinking. Recently, we developed a culture system which guides cells in high-density collagen gels to develop native-like hierarchically organized collagen fibers, which match native fiber alignment and diameters by 6 weeks. However, tensile moduli plateau at 1MPa, suggesting crosslinking may be lacking. Collagen crosslinking is regulated by lysyl oxidase (LOX) which forms immature crosslinks that condense into mature trivalent crosslinks. Trivalent crosslinks are thought to be the primarily source of strength in fibers, but it's not well understood how they form. The objective of this study was to evaluate the effect of exogenous LOX in our culture system at different stages of hierarchical fiber formation to produce stronger replacements and to better understand factors affecting crosslink maturation. We found treatment with LOX isoform LOXL2 did not restrict hierarchical fiber formation, with constructs still forming aligned collagen fibrils by 2 weeks, larger fibers by 4 weeks, and early fascicles by 6 weeks. However, LOXL2 treatment did significantly increase mature pyridinium (PYD) crosslink accumulation and tissue mechanics, with timing of LOXL2 supplementation during fiber formation having a significant effect. Overall, we found one week of LOXL2 supplementation at 4 weeks produced constructs with native-like fiber organization, increased PYD accumulation, and increased mechanics, ultimately matching the tensile modulus of immature bovine menisci. Collagen fibers are the primary source of strength and function in connective tissues throughout the body, however it remains a challenge to develop these fibers in engineered replacements, greatly reducing treatment options. Here we demonstrate lysyl oxidase like 2 (LOXL2) can be used to significantly improve the mechanics of tissue engineered constructs, but timing of application is important and will most likely depend on degree of collagen organization or maturation. Currently there is limited understanding of how collagen crosslinking is regulated, and this system is a promising platform to further investigate cellular regulation of LOX crosslinking. Understanding the mechanism that regulates LOX production and activity is needed to ultimately regenerate functional repair or replacements for connective tissues throughout the body. [Display omitted] [ABSTRACT FROM AUTHOR]

Fibrocartilaginous entheses are structurally complex tissues that translate load from elastic ligaments to stiff bone via complex zonal gradients in the organization, mineralization, and cell phenotype. Currently, these complex gradients necessary for long-term mechanical function are not recreated in soft tissue-to-bone healing or engineered replacements, contributing to high failure rates. Previously, we developed a culture system that guides ligament fibroblasts to develop aligned native-sized collagen fibers using high-density collagen gels and mechanical boundary conditions. These constructs are promising ligament replacements, however functional ligament-to-bone attachments, or entheses, are required for long-term function in vivo. The objective of this study was to investigate the effect of compressive mechanical boundary conditions and the addition of beta-tricalcium phosphate (βTCP), a known osteoconductive agent, on the development of zonal ligament-to-bone entheses. We found that compressive boundary clamps, that restrict cellular contraction and produce a zonal tensile-compressive environment, guide ligament fibroblasts to produce 3 unique zones of collagen organization and zonal accumulation of glycosaminoglycans (GAGs), type II, and type X collagen. Ultimately, by 6 weeks of culture these constructs had similar organization and composition as immature bovine entheses. Further, βTCP applied under the clamp enhanced maturation of these entheses, leading to significantly increased tensile moduli, and zonal GAG accumulation, ALP activity, and calcium-phosphate accumulation, suggesting the initiation of endochondral ossification. This culture system produced some of the most organized entheses to date, closely mirroring early postnatal enthesis development, and provides an in vitro platform to better understand the cues that drive enthesis maturation in vivo. Ligaments are attached to bone via entheses. Entheses are complex tissues with gradients in organization, composition, and cell phenotype. Entheses are necessary for proper transfer of load from ligament-to-bone, but currently are not restored with healing or replacements. Here, we provide new insight into how tensile-compressive boundary conditions and βTCP drive zonal gradients in collagen organization, mineralization, and matrix composition, producing tissues similar to immature ligament-to-bone attachments. Collectively, this culture system uses a bottom-up approach with mechanical and biochemical cues to produce engineered replacements which closely mirror postnatal enthesis development. This culture system is a promising platform to better understanding the cues that regulate enthesis formation so to better drive enthesis regeneration following graft repair and in engineered replacements. [Display omitted] [ABSTRACT FROM AUTHOR]

Partial joint repair is a surgical procedure where an artificial material is used to replace localised chondral damage. These artificial bearing surfaces must articulate against cartilage, but current materials do not replicate both the biphasic and boundary lubrication mechanisms of cartilage. A research challenge therefore exists to provide a material that mimics both boundary and biphasic lubrication mechanisms of cartilage. In this work a polymeric network of a biomimetic boundary lubricant, poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC), was incorporated into an ultra-tough double network (DN) biphasic (water phase + polymer phase) gel, to form a PMPC triple network (PMPC TN) hydrogel with boundary and biphasic lubrication capability. The presence of this third network of MPC was confirmed using ATR-FTIR. The PMPC TN hydrogel had a yield stress of 26 MPa, which is an order of magnitude higher than the peak stresses found in the native human knee. A preliminary pin on plate tribology study was performed where both the DN and PMPC TN hydrogels experienced a reduction in friction with increasing sliding speed which is consistent with biphasic lubrication. In the physiological sliding speed range, the PMPC TN hydrogel halved the friction compared to the DN hydrogel indicating the boundary lubricating PMPC network was working. A biocompatible, tough, strong and chondral lubrication imitating PMPC TN hydrogel was synthesised in this work. By complementing the biphasic and boundary lubrication mechanisms of cartilage, PMPC TN hydrogel could reduce the reported incidence of chondral damage opposite partial joint repair implants, and therefore increase the clinical efficacy of partial joint repair. Statement of Significance This paper presents the synthesis, characterisation and preliminary tribological testing of a new biomaterial that aims to recreate the primary chondral lubrication mechanisms: boundary and biphasic lubrication. This work has demonstrated that the introduction of an established zwitterionic, biomimetic boundary lubricant can improve the frictional properties of an ultra-tough hydrogel. This new biomaterial, when used as a partial joint replacement bearing material, may help avoid damage to the opposing chondral surface—which has been reported as an issue for other non-biomimetic partial joint replacement materials. Alongside the synthesis of a novel biomaterial focused on complementing the lubrication mechanisms of cartilage, your readership will gain insights into effective mechanical and tribological testing methods and materials characterisation methods for their own biomaterials. [ABSTRACT FROM AUTHOR]

Conjugated polymers have been increasingly considered for the design of conductive materials in the field of regenerative medicine. However, optimal scaffold properties addressing the complexity of the desired tissue still need to be developed. The focus of this study lies in the development and evaluation of a conductive scaffold for bone tissue engineering. In this study PEDOT:PSS scaffolds were designed and evaluated in vitro using MC3T3-E1 osteogenic precursor cells, and the cells were assessed for distinct differentiation stages and the expression of an osteogenic phenotype. Ice-templated PEDOT:PSS scaffolds presented high pore interconnectivity with a median pore diameter of 53.6 ± 5.9 µm and a total pore surface area of 7.72 ± 1.7 m 2 ·g −1 . The electrical conductivity, based on I-V curves, was measured to be 140 µS·cm −1 with a reduced, but stable conductivity of 6.1 µS·cm −1 after 28 days in cell culture media. MC3T3-E1 gene expression levels of ALPL, COL1A1 and RUNX2 were significantly enhanced after 4 weeks, in line with increased extracellular matrix mineralisation, and osteocalcin deposition. These results demonstrate that a porous material, based purely on PEDOT:PSS, is suitable as a scaffold for bone tissue engineering and thus represents a promising candidate for regenerative medicine. Statement of Significance Tissue engineering approaches have been increasingly considered for the repair of non-union fractions, craniofacial reconstruction or large bone defect replacements. The design of complex biomaterials and successful engineering of 3-dimensional tissue constructs is of paramount importance to meet this clinical need. Conductive scaffolds, based on conjugated polymers, present interesting candidates to address the piezoelectric properties of bone tissue and to induce enhanced osteogenesis upon implantation. However, conductive scaffolds have not been investigated in vitro in great measure. To this end, we have developed a highly porous, electrically conductive scaffold based on PEDOT:PSS, and provide evidence that this purely synthetic material is a promising candidate for bone tissue engineering. [ABSTRACT FROM AUTHOR]

This study investigated the effect of mechanical anchoring on the development of fiber organization and anisotropy in anatomically shaped tissue engineered menisci. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into molds designed to produce meniscus implants with 12 mm extensions at each horn. After a day of static culture, 10 and 20 mg/ml collagen menisci were either clamped or unclamped and cultured for up to 8 weeks. Clamped menisci were anchored in culture trays throughout culture to mimic the native meniscus horn attachment sites, restrict contraction circumferentially, and encourage circumferential alignment. Clamped menisci retained their size and shape, and by 8 weeks developed circumferential and radial fiber organization that resembled native meniscus. Clamping also increased collagen accumulation and improved mechanical properties compared to unclamped menisci. Enhanced organization in clamped menisci was further reflected in the development of anisotropic tensile properties, with 2-3 fold higher circumferential moduli compared to radial moduli, a similar ratio to native meniscus. Ten and 20 mg/ml clamped menisci had similar levels of organization, with 20 mg/ml menisci producing larger diameter fibers and significantly better mechanical properties. Collectively, these data demonstrate the benefit of using bio-inspired mechanical boundary conditions to drive the formation of a highly organized collagen fiber network. [ABSTRACT FROM AUTHOR]

In this study we analyzed the effects of IGF-I on the boundary lubricating ability of engineered meniscal tissue using a high density collagen gel seeded with meniscal fibrochondrocytes. Biochemical, histological, immunohistochemical, and tribological analyses were carried out to determine a construct's ability to functionally localize lubricin. Our study revealed that supplementation with IGF-I enhanced both the proliferation of cells within the construct as well as enhanced the anabolic activity of the seeded cells. Growth factor supplementation also facilitated the localization of ECM constituents (i.e. fibronectin and type II collagen) near the tissue surface that are important for the localization of lubricin, a boundary lubricant. Consequently, we found localized lubricin in the constructs supplemented with IGF-I. Tribologically, we demonstrated that lubricin serves as a boundary lubricant adsorbed to native meniscal surfaces. Lubricin removal from the native meniscus surface increased boundary friction coefficient by 40%. For the engineered constructs, the lubricin localization facilitated by growth factor supplementation also reduced friction coefficient by a similar margin, but similar results were not evident in control constructs. This study demonstrates that the use of growth factors in meniscal tissue engineering can enhance tribological properties by facilitating the localization of boundary lubricants at the surface of engineered tissue. [ABSTRACT FROM AUTHOR]

Abstract: This study investigates the potential of high density type I collagen gels as an injectable scaffold for tissue engineering of whole menisci, and compares these results with previous strategies using alginate as an injectable scaffold. Bovine meniscal fibrochondrocytes were mixed with collagen and injected into micro-computed tomography-based molds to create 10 and 20mgml−1 menisci that were cultured for up to 4weeks and compared with cultured alginate menisci. Contraction, histological, confocal microscopy, biochemical and mechanical analysis were performed to determine tissue development. After 4weeks culture, collagen menisci had preserved their shape and significantly improved their biochemical and mechanical properties. Both 10 and 20mgml−1 menisci maintained their DNA content while significantly improving the glycosaminoglycan and collagen content, at values significantly higher than the alginate controls. Collagen menisci matched the alginate control in terms of the equilibrium modulus, and developed a 3- to 6-fold higher tensile modulus than alginate by 4weeks. Further fibrochondrocytes were able to reorganize the collagen gels into a more fibrous appearance similar to native menisci. [Copyright &y& Elsevier]

Hierarchical collagen fibers are the primary source of strength in musculoskeletal tendons, ligaments, and menisci. It has remained a challenge to develop these large fibers in engineered replacements or in vivo after injury. The objective of this study was to investigate the ability of restrained cell-seeded high density collagen gels to drive hierarchical fiber formation for multiple musculoskeletal tissues. We found boundary conditions applied to high density collagen gels were capable of driving tenocytes, ligament fibroblasts, and meniscal fibrochondrocytes to develop native-sized hierarchical collagen fibers 20–40 μm in diameter. The fibers organize similar to bovine juvenile collagen with native fibril banding patterns and hierarchical fiber bundles 50–350 μm in diameter by 6 weeks. Mirroring fiber organization, tensile properties of restrained samples improved significantly with time, reaching ~1 MPa. Additionally, tendon, ligament, and meniscal cells produced significantly different sized fibers, different degrees of crimp, and different GAG concentrations, which corresponded with respective juvenile tissue. To our knowledge, these are some of the largest, most organized fibers produced to date in vitro. Further, cells produced tissue specific hierarchical fibers, suggesting this system is a promising tool to better understand cellular regulation of fiber formation to better stimulate it in vivo after injury. Image 1 [ABSTRACT FROM AUTHOR]

Increased wettability of titanium and titanium alloy surfaces due to processing and storage methods increases osteoprogenitor cell differentiation and osseointegration compared to microroughness alone. Implants that are exposed to air have a hydrophobic surface due to adsorption of atmospheric hydrocarbons, which can limit overall implant success. Dielectric barrier discharge plasma (DBD) is one method to increase surface hydrophilicity. Although current DBD methods yield a hydrophilic surface, adsorbed hydrocarbons rapidly restore hydrophobicity. We demonstrated that application of DBD to implants previously packaged in a vacuum, generates a hydrophilic surface that supports osteoblastic differentiation in vitro and this can be done immediately prior to use. In the present study, we tested the hypothesis that DBD treatment to alter surface wettability at the time of implant placement will improve osseointegration in vivo. Twenty male and sixteen female rabbits were used in a preclinical trans-axial femur model of osseointegration. Control and DBD treatment implants were inserted randomized per hind limb in each rabbit (1 implant/hind-limb). At 6 weeks post-surgery, bone-to-implant contact, adjacent bone volume, and torque to failure were assessed by micro-CT, calcified histology, and mechanical testing. DBD plasma treatment of vacuum-sealed implants increased surface wettability and did not change surface chemistry or roughness. Peak torque and torsional energy, and bone-to-implant contact increased with DBD treatment in males. In contrast, female rabbits showed increased osseointegration equal to DBD treated male implants regardless of DBD plasma treatment. DBD treatment is an effective method to enhance osseointegration by increasing surface wettability; however, this response is sex dependent. In healthy female patients, DBD treatment may not be necessary but in older patients or patients with compromised bone, this treatment could be an effective measure to ensure implant success. [ABSTRACT FROM AUTHOR]

In developmental biology, gradients of bioactive signals direct the formation of structural transitions in tissue that are key to physiological function. Failure to reproduce these native features in an in vitro setting can severely limit the success of bioengineered tissue constructs. In this report, we introduce a facile and rapid platform that uses magnetic field alignment of glycosylated superparamagnetic iron oxide nanoparticles, pre-loaded with growth factors, to pattern biochemical gradients into a range of biomaterial systems. Gradients of bone morphogenetic protein 2 in agarose hydrogels were used to spatially direct the osteogenesis of human mesenchymal stem cells and generate robust osteochondral tissue constructs exhibiting a clear mineral transition from bone to cartilage. Interestingly, the smooth gradients in growth factor concentration gave rise to biologically-relevant, emergent structural features, including a tidemark transition demarcating mineralized and non-mineralized tissue and an osteochondral interface rich in hypertrophic chondrocytes. This platform technology offers great versatility and provides an exciting new opportunity for overcoming a range of interfacial tissue engineering challenges. [ABSTRACT FROM AUTHOR]