Characterization of degradation kinetics of additively manufactured PLGA under variable mechanical loading paradigms.

Controlled degradation of biodegradable poly-lactic-co-glycolic acid (PLGA) trauma implants may increase interfragmentary loading which is known to accelerate fracture healing. Additive manufacturing allows us to tune the mechanical properties of PLGA scaffolds; however, little is known about this novel approach. The purpose of this study was to use in vitro and in vivo models to determine the degradative kinetics of additively manufactured test coupons fabricated with PLGA. We hypothesized that 1) increases in infill density would lead to improved initial mechanical properties, and 2) loss of mechanical properties would be constant as a function of time, regardless of implant design. Porous and solid test coupons were fabricated using 85:15 PLGA filament. Coupons were either incubated in serum or implanted subcutaneously in rats for up to 16 weeks. Samples were tested in tension, compression, torsion, and bending on a universal test frame. Variables of interest included, but were not limited to: stiffness, and ultimate force for each unique test. Infill density was the driving factor in test coupon mechanical properties, whereas differences in lattice architecture led to minimal changes. We observed moderate levels of degradation after 8 weeks, and significant decreases for all specimens after 16 weeks. Results from this study suggest substantial degradation of 3-D printed PLGA implants occurs during the 8- to 16-week window, which may be desirable for bone fracture repair applications. This study represents initial findings that will help us better understand the complicated interactions between overall implant design, porosity, and implant biodegradation.

Developmental morphogens direct human induced pluripotent stem cells toward an annulus fibrosus-like cell phenotype.

Introduction: Therapeutic interventions for intervertebral disc herniation remain scarce due to the inability of endogenous annulus fibrosus (AF) cells to respond to injury and drive tissue regeneration. Unlike other orthopedic tissues, such as cartilage, delivery of exogenous cells to the site of annular injury remains underdeveloped, largely due to a lack of an ideal cell source and the invasive nature of cell isolation. Human induced pluripotent stem cells (iPSCs) can be differentiated to specific cell fates using biochemical factors and are, therefore, an invaluable tool for cell therapy approaches. While differentiation protocols have been developed for cartilage and fibrous connective tissues (e.g., tendon), the signals that regulate the induction and differentiation of human iPSCs toward the AF fate remain unknown. Methods: iPSC-derived sclerotome cells were treated with various combinations of developmental signals including transforming growth factor beta 3 (TGF-ß3), connective tissue growth factor (CTGF), platelet derived growth factor BB (PDGF-BB), insulin-like growth factor 1 (IGF-1), or the Hedgehog pathway activator, Purmorphamine, and gene expression changes in major AF-associated ECM genes were assessed. The top performing combination treatments were further validated by using three distinct iPSC lines and by assessing the production of upregulated ECM proteins of interest. To conduct a broader analysis of the transcriptomic shifts elicited by each factor combination, and to compare genetic profiles of treated cells to mature human AF cells, a 96.96 Fluidigm gene expression array was applied, and principal component analysis was employed to identify the transcriptional signatures of each cell population and treatment group in comparison to native AF cells. Results: TGF-ß3, in combination with PDGF-BB, CTGF, or IGF-1, induced an upregulation of key AF ECM genes in iPSC-derived sclerotome cells. In particular, treatment with a combination of TGF-ß3 with PDGF-BB for 14 days significantly increased gene expression of collagen II and aggrecan and increased protein deposition of collagen I and elastin compared to other treatment groups. Assessment of genes uniquely highly expressed by AF cells or SCL cells, respectively, revealed a shift toward the genetic profile of AF cells with the addition of TGF-ß3 and PDGF-BB for 14 days. Discussion: These findings represent an initial approach to guide human induced pluripotent stem cells toward an AF-like fate for cellular delivery strategies.

Tension-activated nanofiber patches delivering an anti-inflammatory drug improve repair in a goat intervertebral disc herniation model.

Conventional microdiscectomy treatment for intervertebral disc herniation alleviates pain but does not repair the annulus fibrosus, resulting in a high incidence of recurrent herniation and persistent dysfunction. The lack of repair and the acute inflammation that arise after injury can further compromise the disc and result in disc-wide degeneration in the long term. To address this clinical need, we developed tension-activated repair patches (TARPs) for annulus fibrosus repair and local delivery of the anti-inflammatory factor anakinra (a recombinant interleukin-1 receptor antagonist). TARPs transmit physiologic strain to mechanically activated microcapsules embedded within the patch, which release encapsulated bioactive molecules in direct response to spinal loading. Mechanically activated microcapsules carrying anakinra were loaded into TARPs, and the effects of TARP-mediated annular repair and anakinra delivery were evaluated in a goat model of annular injury in the cervical spine. TARPs integrated with native tissue and provided structural reinforcement at the injury site that prevented aberrant disc-wide remodeling resulting from detensioning of the annular fibrosus. The delivery of anakinra by TARP implantation increased matrix deposition and retention at the injury site and improved maintenance of disc extracellular matrix. Anakinra delivery additionally attenuated the inflammatory response associated with TARP implantation, decreasing osteolysis in adjacent vertebrae and preserving disc cellularity and matrix organization throughout the annulus fibrosus. These results demonstrate the therapeutic potential of TARPs for the treatment of intervertebral disc herniation.

Gravity-based patterning of osteogenic factors to preserve bone structure after osteochondral injury in a large animal model.

Chondral and osteochondral repair strategies are limited by adverse bony changes that occur after injury. Bone resorption can cause entire scaffolds, engineered tissues, or even endogenous repair tissues to subside below the cartilage surface. To address this translational issue, we fabricated thick-shelled poly(D,L-lactide-co-glycolide) microcapsules containing the pro-osteogenic agents triiodothyronine andß-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that the developed microcapsules rupturedin vitrounder increasing mechanical loads, and readily sink within a liquid solution, enabling gravity-based patterning along the osteochondral surface. In a large animal, these mechanically-activated microcapsules (MAMCs) were assessed through two different delivery strategies. Intra-articular injection of control MAMCs enabled fluorescent quantification of MAMC rupture and cargo release in a synovial joint setting over timein vivo. This joint-wide injection also confirmed that the MAMCs do not elicit an inflammatory response. In the contralateral hindlimbs, chondral defects were created, MAMCs were patternedin situ, and nanofracture (Nfx), a clinically utilized method to promote cartilage repair, was performed. The Nfx holes enabled marrow-derived stromal cells to enter the defect area and served as repeatable bone injury sites to monitor over time. Animals were evaluated one and two weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over two weeks. A bone fluorochrome label injected at the time of surgery displayed maintenance of mineral labeling in the therapeutic group, but resorption in both control groups. Alkaline phosphatase (AP) staining at the osteochondral interface revealed higher AP activity in defects treated with therapeutic MAMCs. Overall, this study develops a gravity-based approach to pattern bioactive factors along the osteochondral interface, and applies this novel biofabrication strategy to preserve bone structure after osteochondral injury.

Disrupted Surfaces of Porous Membranes Reduce Nuclear YAP Localization and Enhance Adipogenesis through Morphological Changes.

The disrupted surface of porous membranes, commonly used in tissue-chip and cellular coculture systems, is known to weaken cell-substrate interactions. Here, we investigated whether disrupted surfaces of membranes with micron and submicron scale pores affect yes-associated protein (YAP) localization and differentiation of adipose-derived stem cells. We found that these substrates reduce YAP nuclear localization through decreased cell spreading, consistent with reduced cell-substrate interactions, and in turn enhance adipogenesis while decreasing osteogenesis.

Stretch-responsive adhesive microcapsules for strain-regulated antibiotic release from fabric wound dressings.

Bacterial infection of a wound is a major complication that can significantly delay proper healing and even necessitate surgical debridement. Conventional non-woven fabric dressings, including gauzes, bandages and cotton wools, often fail in treating wound infections in a timely manner due to their passive release mechanism of antibiotics. Here, we propose adhesive mechanically-activated microcapsules (MAMCs) capable of strongly adhering to a fibrous matrix to achieve a self-regulated release of antibiotics upon uniaxial stretching of non-woven fabric dressings. To achieve this, a uniform population of polydopamine (PDA)-coated MAMCs (PDA-MAMCs) are prepared using a microfluidics technique and subsequent oxidative dopamine polymerization. The PDA-MAMC allows for robust mechano-activation within the fibrous network through high retention and effective transmission of mechanical force under stretching. By validating the potential of a PDA-MAMCs-laden gauze to release antibiotics in a tensile strain-dependent manner, we demonstrate that PDA-MAMCs can be successfully incorporated into a woven material and create a smart wound dressing for control of bacterial infections. This new mechano-activatable delivery approach will open up a new avenue for a stretch-triggered, on-demand release of therapeutic cargos in skin-mountable or wearable biomedical devices.

A challenging playing field: Identifying the endogenous impediments to annulus fibrosus repair.

Intervertebral disc (IVD) herniations, caused by annulus fibrosus (AF) tears that enable disc tissue extrusion beyond the disc space, are very prevalent, especially among adults in the third to fifth decade of life. Symptomatic herniations, in which the extruded tissue compresses surrounding nerves, are characterized by back pain, numbness, and tingling and can cause extreme physical disability. Patients whose symptoms persist after nonoperative intervention may undergo surgical removal of the herniated tissue via microdiscectomy surgery. The AF, however, which has a poor endogenous healing ability, is left unrepaired increasing the risk for re-herniation and pre-disposing the IVD to degenerative disc disease. The lack of understanding of the mechanisms involved in native AF repair limits the design of repair systems that overcome the impediments to successful AF restoration. Moreover, the complexity of the AF structure and the challenging anatomy of the repair environment represents a significant challenge for the design of new repair devices. While progress has been made towards the development of an effective AF repair technique, these methods have yet to demonstrate long-term repair and recovery of IVD biomechanics. In this review, the limitations of endogenous AF healing are discussed and key cellular events and factors involved are highlighted to identify potential therapeutic targets that can be integrated into AF repair methods. Clinical repair strategies and their limitations are described to further guide the design of repair approaches that effectively restore native tissue structure and function.

Putting the Pieces in Place: Mobilizing Cellular Players to Improve Annulus Fibrosus Repair.

The intervertebral disc (IVD) is an integral load-bearing tissue that derives its function from its composite structure and extracellular matrix composition. IVD herniations involve the failure of the annulus fibrosus (AF) and the extrusion of the nucleus pulposus beyond the disc boundary. Disc herniations can impinge the neural elements and cause debilitating pain and loss of function, posing a significant burden on individual patients and society as a whole. Patients with persistent symptoms may require surgery; however, surgical intervention fails to repair the ruptured AF and is associated with the risk for reherniation and further disc degeneration. Given the limitations of AF endogenous repair, many attempts have been made toward the development of effective repair approaches that reestablish IVD function. These methods, however, fail to recapitulate the composition and organization of the native AF, ultimately resulting in inferior tissue mechanics and function over time and high rates of reherniation. Harnessing the cellular function of cells (endogenous or exogenous) at the repair site through the provision of cell-instructive cues could enhance AF tissue regeneration and, ultimately, improve healing outcomes. In this study, we review the diverse approaches that have been developed for AF repair and emphasize the potential for mobilizing the appropriate cellular players at the site of injury to improve AF healing. Impact statement Conventional treatments for intervertebral disc herniation fail to repair the annulus fibrosus (AF), increasing the risk for recurrent herniation. The lack of repair devices in the market has spurred the development of regenerative approaches, yet most of these rely on a scarce endogenous cell population to repair large injuries, resulting in inadequate regeneration. This review identifies current and developing strategies for AF repair and highlights the potential for harnessing cellular function to improve AF regeneration. Ideal cell sources, differentiation strategies, and delivery methods are discussed to guide the design of repair systems that leverage specialized cells to achieve superior outcomes.

Mechano-activated biomolecule release in regenerating load-bearing tissue microenvironments.

Although mechanical loads are integral for musculoskeletal tissue homeostasis, overloading and traumatic events can result in tissue injury. Conventional drug delivery approaches for musculoskeletal tissue repair employ localized drug injections. However, rapid drug clearance and inadequate synchronization of molecule provision with healing progression render these methods ineffective. To overcome this, a programmable mechanoresponsive drug delivery system was developed that utilizes the mechanical environment of the tissue during rehabilitation (for example, during cartilage repair) to trigger biomolecule provision. For this, a suite of mechanically-activated microcapsules (MAMCs) with different rupture profiles was generated in a single fabrication batch via osmotic annealing of double emulsions. MAMC physical dimensions were found to dictate mechano-activation in 2D and 3D environments and their stability in vitro and in vivo, demonstrating the tunability of this system. In models of cartilage regeneration, MAMCs did not interfere with tissue growth and activated depending on the mechanical properties of the regenerating tissue. Finally, biologically active anti-inflammatory agents were encapsulated and released from MAMCs, which counteracted degradative cues and prevented the loss of matrix in living tissue environments. This unique technology has tremendous potential for implementation across a wide array of musculoskeletal conditions for enhanced repair of load-bearing tissues.

Nuclear softening expedites interstitial cell migration in fibrous networks and dense connective tissues.

Dense matrices impede interstitial cell migration and subsequent repair. We hypothesized that nuclear stiffness is a limiting factor in migration and posited that repair could be expedited by transiently decreasing nuclear stiffness. To test this, we interrogated the interstitial migratory capacity of adult meniscal cells through dense fibrous networks and adult tissue before and after nuclear softening via the application of a histone deacetylase inhibitor, Trichostatin A (TSA) or knockdown of the filamentous nuclear protein Lamin A/C. Our results show that transient softening of the nucleus improves migration through microporous membranes, electrospun fibrous matrices, and tissue sections and that nuclear properties and cell function recover after treatment. We also showed that biomaterial delivery of TSA promoted in vivo cellularization of scaffolds by endogenous cells. By addressing the inherent limitations to repair imposed by nuclear stiffness, this work defines a new strategy to promote the repair of damaged dense connective tissues.

Influence of Fiber Stiffness on Meniscal Cell Migration into Dense Fibrous Networks.

Fibrous scaffolds fabricated via electrospinning are being explored to repair injuries within dense connective tissues. However, there is still much to be understood regarding the appropriate scaffold properties that best support tissue repair. In this study, the influence of the stiffness of electrospun fibers on cell invasion into fibrous scaffolds is investigated. Specifically, soft and stiff electrospun fibrous networks are fabricated from crosslinked methacrylated hyaluronic acid (MeHA), where the stiffness is altered via the extent of MeHA crosslinking. Meniscal fibrochondrocyte (MFC) adhesion and migration into fibrous networks are investigated, where the softer MeHA fibrous networks are easily deformed and densified through cellular tractions and the stiffer MeHA fibrous networks support ≈50% greater MFC invasion over weeks when placed adjacent to meniscal tissue. When the scaffolds are sandwiched between meniscal tissues and implanted subcutaneously, the stiffer MeHA fibrous networks again support enhanced cellular invasion and greater collagen deposition after 4 weeks when compared to the softer MeHA fibrous networks. These results indicate that the mechanics and deformability of fibrous networks likely alter cellular interactions and invasion, providing an important design parameter toward the engineering of scaffolds for tissue repair.

Mechanosensing at Cellular Interfaces.

At the plasma membrane interface, cells use various adhesions to sense their extracellular environment. These adhesions facilitate the transmission of mechanical signals that dictate cell behavior. This review discusses the mechanisms by which these mechanical signals are transduced through cell-matrix and cell-cell adhesions and how this mechanotransduction influences cell processes. Cell-matrix adhesions require the activation of and communication between various transmembrane protein complexes such as integrins. These links at the plasma membrane affect how a cell senses and responds to its matrix environment. Cells also communicate with each other through cell-cell adhesions, which further regulate cell behavior on a single- and multicellular scale. Coordination and competition between cell-cell and cell-matrix adhesions in multicellular aggregates can, to a significant extent, be modeled by differential adhesion analyses between the different interfaces even without knowing the details of cellular signaling. In addition, cell-matrix and cell-cell adhesions are connected by an intracellular cytoskeletal network that allows for direct communication between these distinct adhesions and activation of specific signaling pathways. Other membrane-embedded protein complexes, such as growth factor receptors and ion channels, play additional roles in mechanotransduction. Overall, these mechanoactive elements show the dynamic interplay between the cell, its matrix, and neighboring cells and how these relationships affect cellular function.

Membrane Pore Spacing Can Modulate Endothelial Cell-Substrate and Cell-Cell Interactions.

Mechanical cues and substrate interaction affect the manner in which cells adhere, spread, migrate and form tissues. With increased interest in tissue-on-a-chip and co-culture systems utilizing porous membranes, it is important to understand the role of disrupted surfaces on cellular behavior. Using a transparent glass membrane with defined pore geometries, we investigated endothelial fibronectin fibrillogenesis and formation of focal adhesions as well as development of intercellular junctions. Cells formed fewer focal adhesions and had shorter fibronectin fibrils on porous membranes compared to non-porous controls, which was similar to cell behavior on continuous soft substrates with Young's moduli seven orders of magnitude lower than glass. Additionally, porous membranes promoted enhanced cell-cell interactions as evidenced by earlier formation of tight junctions. These findings suggest that porous membranes with discontinuous surfaces promote reduced cell-matrix interactions similarly to soft substrates and may enhance tissue and barrier formation.

Effect of Simplified One-Step Drilling Protocol on Osseointegration.

This study was designed to compare the combined effect of two different drilling techniques (conventional expansion and one-step) and four different implant geometries in a beagle dog model. The nondecalcified bone-implant samples underwent histologic/metric analysis at 2 and 6 weeks. Morphologic analysis showed similarities between different drilling technique groups and implant geometries. Histomorphometric parameters, bone-to-implant contact (BIC), and bone area fraction occupancy (BAFO) were analyzed, and no statistical difference between drilling groups and/or implant geometry was found. Time was the only variable that affected BIC and BAFO, suggesting that the two protocols are equally biocompatible and osseoconductive.