Neonatal Spinal Cord Transection Decreases Hindlimb Weight-Bearing and Affects Formation of Achilles and Tail Tendons.

Mechanical loading may be required for proper tendon formation. However, it is not well understood how tendon formation is impacted by the development of weight-bearing locomotor activity in the neonate. This study assessed tendon mechanical properties, and concomitant changes in weight-bearing locomotion, in neonatal rats subjected to a low thoracic spinal cord transection or a sham surgery at postnatal day (P)1. On P10, spontaneous locomotion was evaluated in spinal cord transected and sham controls to determine impacts on weight-bearing hindlimb movement. The mechanical properties of P10 Achilles tendons (ATs), as representative energy-storing, weight-bearing tendons, and tail tendons (TTs), as representative positional, non-weight-bearing tendons were evaluated. Non- and partial weight-bearing hindlimb activity decreased in spinal cord transected rats compared to sham controls. No spinal cord transected rats showed full weight-bearing locomotion. ATs from spinal cord transected rats had increased elastic modulus, while cross-sectional area trended lower compared to sham rats. TTs from spinal cord transected rats had higher stiffness and cross-sectional area. Collagen structure of ATs and TTs did not appear impacted by surgery condition, and no significant differences were detected in the collagen crimp pattern. Our findings suggest that mechanical loading from weight-bearing locomotor activity during development regulates neonatal AT lateral expansion and maintains tendon compliance, and that TTs may be differentially regulated. The onset and gradual increase of weight-bearing movement in the neonate may provide the mechanical loading needed to direct functional postnatal tendon formation.

TGFß2-induced tenogenesis impacts cadherin and connexin cell-cell junction proteins in mesenchymal stem cells.

Tenogenic differentiation of stem cells is needed for tendon tissue engineering approaches. A current challenge is the limited information on the cellular-level changes during tenogenic induction. Tendon cells in embryonic and adult tendons possess an array of cell-cell junction proteins that include cadherins and connexins, but how these proteins are impacted by tenogenic differentiation is unknown. Our objective was to explore how tenogenic induction of mesenchymal stem cells (MSCs) using the transforming growth factor (TGF)ß2 impacted protein markers of tendon differentiation and protein levels of N-cadherin, cadherin-11 and connexin-43. MSCs were treated with TGFß2 for 21 days. At 3 days, TGFß2-treated MSCs developed a fibroblastic morphology and significantly decreased levels of N-cadherin protein, which were maintained through 21 days. Similar decreases in protein levels were found for cadherin-11. Connexin-43 protein levels significantly increased at 3 days, but then decreased below control levels, though not significantly. Protein levels of scleraxis and tenomodulin were significantly increased at day 14 and 21, respectively. Taken together, our results indicate that TGFß2 is an inducer of tendon marker proteins (scleraxis and tenomodulin) in MSCs and that tenogenesis alters the protein levels of N-cadherin, cadherin-11 and connexin-43. These findings suggest a role for connexin-43 early in tenogenesis, and show that early-onset and sustained decreases in N-cadherin and cadherin-11 may be novel markers of tenogenesis in MSCs.

Engineering Cell-ECM-Material Interactions for Musculoskeletal Regeneration.

The extracellular microenvironment regulates many of the mechanical and biochemical cues that direct musculoskeletal development and are involved in musculoskeletal disease. The extracellular matrix (ECM) is a main component of this microenvironment. Tissue engineered approaches towards regenerating muscle, cartilage, tendon, and bone target the ECM because it supplies critical signals for regenerating musculoskeletal tissues. Engineered ECM-material scaffolds that mimic key mechanical and biochemical components of the ECM are of particular interest in musculoskeletal tissue engineering. Such materials are biocompatible, can be fabricated to have desirable mechanical and biochemical properties, and can be further chemically or genetically modified to support cell differentiation or halt degenerative disease progression. In this review, we survey how engineered approaches using natural and ECM-derived materials and scaffold systems can harness the unique characteristics of the ECM to support musculoskeletal tissue regeneration, with a focus on skeletal muscle, cartilage, tendon, and bone. We summarize the strengths of current approaches and look towards a future of materials and culture systems with engineered and highly tailored cell-ECM-material interactions to drive musculoskeletal tissue restoration. The works highlighted in this review strongly support the continued exploration of ECM and other engineered materials as tools to control cell fate and make large-scale musculoskeletal regeneration a reality.

Sudan Black B Pretreatment to Suppress Autofluorescence in Silk Fibroin Scaffolds.

Natural polymers are extensively utilized as scaffold materials in tissue engineering and 3D disease modeling due to their general features of cytocompatibility, biodegradability, and ability to mimic the architecture and mechanical properties of the native tissue. A major limitation of many polymeric scaffolds is their autofluorescence under common imaging methods. This autofluorescence, a particular challenge with silk fibroin materials, can interfere with the visualization of fluorescently labeled cells and proteins grown on or in these scaffolds, limiting the assessment of outcomes. Here, Sudan Black B (SBB) was successfully used prefixation prior to cell seeding, in various silk matrices and 3D model systems to quench silk autofluorescence for live cell imaging. SBB was also trialed postfixation in silk hydrogels. We validated that multiple silk scaffolds pretreated with SBB (hexafluoro-2-propanol-silk scaffolds, salt-leached sponges, gel-spun catheters, and sponge-gel composite scaffolds) cultured with fibroblasts, adipose tissue, neural cells, and myoblasts demonstrated improved image resolution when compared to the nonpretreated scaffolds, while also maintaining normal cell behavior (attachment, growth, proliferation, differentiation). SBB pretreatment of silk scaffolds is an option for scaffold systems that require autofluorescence suppression.

The LINC complex regulates Achilles tendon elastic modulus, Achilles and tail tendon collagen crimp, and Achilles and tail tendon lateral expansion during early postnatal development.

There is limited understanding of how mechanical signals regulate tendon development. The nucleus has emerged as a major regulator of cellular mechanosensation, via the linker of nucleoskeleton and cytoskeleton (LINC) protein complex. Specific roles of LINC in tenogenesis have not been explored. In this study, we investigate how LINC regulates tendon development by disabling LINC-mediated mechanosensing via dominant negative (dn) expression of the Klarsicht, ANC-1, and Syne Homology (KASH) domain, which is necessary for LINC to function. We hypothesized that LINC regulates mechanotransduction in developing tendon, and that disabling LINC would impact tendon mechanical properties and structure in a mouse model of dnKASH. We used Achilles (AT) and tail (TT) tendons as representative energy-storing and limb-positioning tendons, respectively. Mechanical testing at postnatal day 10 showed that disabling the LINC complex via dnKASH significantly impacted tendon mechanical properties and cross-sectional area, and that effects differed between ATs and TTs. Collagen crimp distance was also impacted in dnKASH tendons, and was significantly decreased in ATs, and increased in TTs. Overall, we show that disruption to the LINC complex specifically impacts tendon mechanics and collagen crimp structure, with unique responses between an energy-storing and limb-positioning tendon. This suggests that nuclear mechanotransduction through LINC plays a role in regulating tendon formation during neonatal development.

Localized, on-demand, sustained drug delivery from biopolymer-based materials.

INTRODUCTION: Local drug delivery facilitiates higher concentrations of drug molecules at or near the treatment site to enhance treatment efficiency and reduce drug toxicity and other systemic side effects. However, local drug delivery systems face challenges in terms of encapsulation, delivery, and controlled release of therapeutics. AREAS COVERED: We provide an overview of naturally derived biopolymer-based drug delivery systems for localized, sustained, and on-demand treatment. We introduce the advantages and limitations of these systems for drug encapsulation, delivery, and local release, as well as recent applications. EXPERT OPINION: Naturally derived biopolymers like cellulose, silk fibroin, chitosan, alginate, hyaluronic acid, and gelatin are good candidates for localized drug delivery because they are readily chemically modified, biocompatible, biodegradable (with the generation of metabolically compatible degradation products), and can be processed in aqueous and ambient environments to maintain the bioactivity of various therapeutics. The tradeoff between the effective treatment dosage and the response by local healthy tissue should be balanced during the design of these delivery systems. Future directions will be focused on strategies to design tunable and controlled biodegradation rates, as well as to explore commercial utility in substituting biopolymer-based systems for currently utilized synthetic polymers for implants for drug delivery.

Perspectives on scaling production of adipose tissue for food applications.

With rising global demand for food proteins and significant environmental impact associated with conventional animal agriculture, it is important to develop sustainable alternatives to supplement existing meat production. Since fat is an important contributor to meat flavor, recapitulating this component in meat alternatives such as plant based and cell cultured meats is important. Here, we discuss the topic of cell cultured or tissue engineered fat, growing adipocytes in vitro that could imbue meat alternatives with the complex flavor and aromas of animal meat. We outline potential paths for the large scale production of in vitro cultured fat, including adipogenic precursors during cell proliferation, methods to adipogenically differentiate cells at scale, as well as strategies for converting differentiated adipocytes into 3D cultured fat tissues. We showcase the maturation of knowledge and technology behind cell sourcing and scaled proliferation, while also highlighting that adipogenic differentiation and 3D adipose tissue formation at scale need further research. We also provide some potential solutions for achieving adipose cell differentiation and tissue formation at scale based on contemporary research and the state of the field.

Dielectrophoretic Characterization of Tenogenically Differentiating Mesenchymal Stem Cells.

Tendons are collagenous musculoskeletal tissues that connect muscles to bones and transfer the forces necessary for movement. Tendons are susceptible to injury and heal poorly, with long-term loss of function. Mesenchymal stem cell (MSC)-based therapies are a promising approach for treating tendon injuries but are challenged by the difficulties of controlling stem cell fate and of generating homogenous populations of stem cells optimized for tenogenesis (differentiation toward tendon). To address this issue, we aim to explore methods that can be used to identify and ultimately separate tenogenically differentiated MSCs from non-tenogenically differentiated MSCs. In this study, baseline and tenogenically differentiating murine MSCs were characterized for dielectric properties (conductivity and permittivity) of their outer membrane and cytoplasm using a dielectrophoretic (DEP) crossover technique. Experimental results showed that unique dielectric properties distinguished tenogenically differentiating MSCs from controls after three days of tenogenic induction. A single shell model was used to quantify the dielectric properties and determine membrane and cytoplasm conductivity and permittivity. Together, cell responses at the crossover frequency, cell morphology, and shell models showed that changes potentially indicative of early tenogenesis could be detected in the dielectric properties of MSCs as early as three days into differentiation. Differences in dielectric properties with tenogenesis indicate that the DEP-based label-free separation of tenogenically differentiating cells is possible and avoids the complications of current label-dependent flow cytometry-based separation techniques. Overall, this work illustrates the potential of DEP to generate homogeneous populations of differentiated stem cells for applications in tissue engineering and regenerative medicine.

Akt signaling is activated by TGFß2 and impacts tenogenic induction of mesenchymal stem cells.

BACKGROUND: Tissue engineered and regenerative approaches for treating tendon injuries are challenged by the limited information on the cellular signaling pathways driving tenogenic differentiation of stem cells. Members of the transforming growth factor (TGF) ß family, particularly TGFß2, play a role in tenogenesis, which may proceed via Smad-mediated signaling. However, recent evidence suggests some aspects of tenogenesis may be independent of Smad signaling, and other pathways potentially involved in tenogenesis are understudied. Here, we examined the role of Akt/mTORC1/P70S6K signaling in early TGFß2-induced tenogenesis of mesenchymal stem cells (MSCs) and evaluated TGFß2-induced tenogenic differentiation when Smad3 is inhibited. METHODS: Mouse MSCs were treated with TGFß2 to induce tenogenesis, and Akt or Smad3 signaling was chemically inhibited using the Akt inhibitor, MK-2206, or the Smad3 inhibitor, SIS3. Effects of TGFß2 alone and in combination with these inhibitors on the activation of Akt signaling and its downstream targets mTOR and P70S6K were quantified using western blot analysis, and cell morphology was assessed using confocal microscopy. Levels of the tendon marker protein, tenomodulin, were also assessed. RESULTS: TGFß2 alone activated Akt signaling during early tenogenic induction. Chemically inhibiting Akt prevented increases in tenomodulin and attenuated tenogenic morphology of the MSCs in response to TGFß2. Chemically inhibiting Smad3 did not prevent tenogenesis, but appeared to accelerate it. MSCs treated with both TGFß2 and SIS3 produced significantly higher levels of tenomodulin at 7 days and morphology appeared tenogenic, with localized cell alignment and elongation. Finally, inhibiting Smad3 did not appear to impact Akt signaling, suggesting that Akt may allow TGFß2-induced tenogenesis to proceed during disruption of Smad3 signaling. CONCLUSIONS: These findings show that Akt signaling plays a role in TGFß2-induced tenogenesis and that tenogenesis of MSCs can be initiated by TGFß2 during disruption of Smad3 signaling. These findings provide new insights into the signaling pathways that regulate tenogenic induction in stem cells.

Cell-cell junctions in developing and adult tendons.

Tendons connect muscles to bones to transfer the forces necessary for movement. Cell-cell junction proteins, cadherins and connexins, may play a role in tendon development and injury. In this review, we begin by highlighting current understanding of how cell-cell junctions may regulate embryonic tendon development and differentiation. We then examine cell-cell junctions in postnatal tendon, before summarizing the role of cadherins and connexins in adult tendons. More information exists regarding the role of cell-cell junctions in the formation and homeostasis of other musculoskeletal tissues, namely cartilage and bone. Therefore, to inform future tendon studies, we include a brief survey of cadherins and connexins in chondrogenesis and osteogenesis, and summarize how cell-cell junctions are involved in some musculoskeletal tissue pathologies. An enhanced understanding of how cell-cell junctions participate in tendon development, maintenance, and disease will benefit future regenerative strategies.

Dielectrophoresis as a tool for electrophysiological characterization of stem cells.

Dielectrophoresis (DEP), a nonlinear electrokinetic technique caused by Maxwell-Wagner interfacial polarization of neutral particles in an electrolyte solution, is a powerful cell manipulation method used widely for various applications such as enrichment, trapping, and sorting of heterogeneous cell populations. While conventional cell characterization and sorting methods require tagging or labeling of cells, DEP has the potential to manipulate cells in a label-free way. Due to its unique ability to characterize and sort cells without the need of labeling, there is renewed interest in using DEP for stem cell research and regenerative medicine. Stem cells have the potential to differentiate into various lineages, but achieving homogeneous cell phenotypes from an initially heterogeneous cell population is a challenge. Using DEP to efficiently and affordably identify, sort, and enrich either undifferentiated or differentiated stem cell populations in a label-free way would advance their potential uses for applications in tissue engineering and regenerative medicine. This review summarizes recent, significant research findings regarding the electrophysiological characterization of stem cells, with a focus on cellular dielectric properties, i.e., permittivity and conductivity, and on studies that have obtained these measurements using techniques that preserve cell viability, such as crossover frequency. Potential applications for DEP in regenerative medicine are also discussed. Overall, DEP is a promising technique and, when used to characterize, sort, and enrich stem cells, will advance stem cell-based regenerative therapies.

Ex-vivo quantification of ovine pia arachnoid complex biomechanical properties under uniaxial tension.

BACKGROUND: The pia arachnoid complex (PAC) is a cerebrospinal fluid-filled tissue conglomerate that surrounds the brain and spinal cord. Pia mater adheres directly to the surface of the brain while the arachnoid mater adheres to the deep surface of the dura mater. Collagen fibers, known as subarachnoid trabeculae (SAT) fibers, and microvascular structure lie intermediately to the pia and arachnoid meninges. Due to its structural role, alterations to the biomechanical properties of the PAC may change surface stress loading in traumatic brain injury (TBI) caused by sub-concussive hits. The aim of this study was to quantify the mechanical and morphological properties of ovine PAC. METHODS: Ovine brain samples (n = 10) were removed from the skull and tissue was harvested within 30 min post-mortem. To access the PAC, ovine skulls were split medially from the occipital region down the nasal bone on the superior and inferior aspects of the skull. A template was used to remove arachnoid samples from the left and right sides of the frontal and occipital regions of the brain. 10 ex-vivo samples were tested with uniaxial tension at 2 mm s-1, average strain rate of 0.59 s-1, until failure at < 5 h post extraction. The force and displacement data were acquired at 100 Hz. PAC tissue collagen fiber microstructure was characterized using second-harmonic generation (SHG) imaging on a subset of n = 4 stained tissue samples. To differentiate transverse blood vessels from SAT by visualization of cell nuclei and endothelial cells, samples were stained with DAPI and anti-von Willebrand Factor, respectively. The Mooney-Rivlin model for average stress-strain curve fit was used to model PAC material properties. RESULTS: The elastic modulus, ultimate stress, and ultimate strain were found to be 7.7 ± 3.0, 2.7 ± 0.76 MPa, and 0.60 ± 0.13, respectively. No statistical significance was found across brain dissection locations in terms of biomechanical properties. SHG images were post-processed to obtain average SAT fiber intersection density, concentration, porosity, tortuosity, segment length, orientation, radial counts, and diameter as 0.23, 26.14, 73.86%, 1.07 ± 0.28, 17.33 ± 15.25 µm, 84.66 ± 49.18°, 8.15%, 3.46 ± 1.62 µm, respectively. CONCLUSION: For the sizes, strain, and strain rates tested, our results suggest that ovine PAC mechanical behavior is isotropic, and that the Mooney-Rivlin model is an appropriate curve-fitting constitutive equation for obtaining material parameters of PAC tissues.

Models of tendon development and injury.

Tendons link muscle to bone and transfer forces necessary for normal movement. Tendon injuries can be debilitating and their intrinsic healing potential is limited. These challenges have motivated the development of model systems to study the factors that regulate tendon formation and tendon injury. Recent advances in understanding of embryonic and postnatal tendon formation have inspired approaches that aimed to mimic key aspects of tendon development. Model systems have also been developed to explore factors that regulate tendon injury and healing. We highlight current model systems that explore developmentally inspired cellular, mechanical, and biochemical factors in tendon formation and tenogenic stem cell differentiation. Next, we discuss in vivo, in vitro, ex vivo, and computational models of tendon injury that examine how mechanical loading and biochemical factors contribute to tendon pathologies and healing. These tendon development and injury models show promise for identifying the factors guiding tendon formation and tendon pathologies, and will ultimately improve regenerative tissue engineering strategies and clinical outcomes.

Onset of neonatal locomotor behavior and the mechanical development of Achilles and tail tendons.

Tendon tissue engineering approaches are challenged by a limited understanding of the role mechanical loading plays in normal tendon development. We propose that the increased loading that developing postnatal tendons experience with the onset of locomotor behavior impacts tendon formation. The objective of this study was to assess the onset of spontaneous weight-bearing locomotion in postnatal day (P) 1, 5, and 10 rats, and characterize the relationship between locomotion and the mechanical development of weight-bearing and non-weight-bearing tendons. Movement was video recorded and scored to determine non-weight-bearing, partial weight-bearing, and full weight-bearing locomotor behavior at P1, P5, and P10. Achilles tendons, as weight-bearing tendons, and tail tendons, as non-weight-bearing tendons, were mechanically evaluated. We observed a significant increase in locomotor behavior in P10 rats, compared to P1 and P5. We also found corresponding significant differences in the maximum force, stiffness, displacement at maximum force, and cross-sectional area in Achilles tendons, as a function of postnatal age. However, the maximum stress, strain at maximum stress, and elastic modulus remained constant. Tail tendons of P10 rats had significantly higher maximum force, maximum stress, elastic modulus, and stiffness compared to P5. Our results suggest that the onset of locomotor behavior may be providing the mechanical cues regulating postnatal tendon growth, and their mechanical development may proceed differently in weight-bearing and non-weight-bearing tendons. Further analysis of how this loading affects developing tendons in vivo may inform future engineering approaches aiming to apply such mechanical cues to regulate engineered tendon formation in vitro.

A 3D printed mechanical bioreactor for investigating mechanobiology and soft tissue mechanics.

Mechanical loading is an important cue for directing stem cell fate and engineered tissue formation in vitro. Stem cells cultured on 2-dimensional (D) substrates and in 3D scaffolds have been shown to differentiate toward bone, tendon, cartilage, ligament, and skeletal muscle lineages depending on their exposure to mechanical stimuli. To apply this mechanical stimulus in vitro, mechanical bioreactors are needed. However, current bioreactor systems are challenged by their high cost, limited ability for customization, and lack of force measurement capabilities. We demonstrate the use of 3-dimensional printing (3DP) technology to design and fabricate a low-cost custom bioreactor system that can be used to apply controlled mechanical stimuli to cells in culture and measure the mechanical properties of small soft tissues. The results of our in vitro studies and mechanical evaluations show that 3DP technology is feasible as a platform for developing a low-cost, customizable, and multifunctional mechanical bioreactor system. • 3DP technology was used to print a multifunctional bioreactor system/tensile load frame for a fraction of the cost of commercial systems. • The system mechanically stimulated cells in culture and evaluated the mechanical properties of soft tissues. • This system is easily customizable and can be used to evaluate multiple types of soft tissues.