Biology subjects

Locke, R. C.

Publications and source records attributed to Locke, R. C..

10 recordsLinked to original sources

bioRxiv2022

Rapid Restoration of Cell Phenotype and Matrix Forming Capacity Following Transient Nuclear Softening

The dense extracellular matrix of connective tissues impedes cell migration and subsequent matrix formation at sites of injury. We recently employed transient nuclear softening via histone deacetylase inhibition with trichostatin A (TSA) treatment to overcome the stiff nuclear impediments to cell migration through dense tissues and electrospun matrices. Despite these positive findings, the long-term implications of transient nuclear softening on cell transcriptional phenotype and matrix formation capacity are unknown. To address this, we investigated the influence of transient TSA treatment on porcine meniscal cell behavior, beginning with the efficacy and reproducibility of transient TSA treatment on histone acetylation and chromatin remodeling in vitro and cell migration through native meniscus tissue. Within 3 days after cessation of transient TSA treatment, histone acetylation and chromatin remodeling returned to control levels. Following TSA treatment, endogenous cell migration through native meniscus tissue increased greater than 3-fold compared to controls. Importantly, meniscal cells completely restored their transcriptional phenotype and maintained their capacity to respond transcriptionally and functionally to a secondary pro-matrix stimuli (i.e., transforming growth factor {beta}3) within 7 days after cessation of TSA treatment. Towards translation, we also showed the feasibility of biomaterial-delivered TSA to increase endogenous cell migration to a wound edge ex vivo. Together, this work defines the efficacy, reproducibility, safety, and feasibility of future translational approaches for nuclear softening to treat dense connective tissue injuries.

genomics↗

bioRxiv2022

Mechano-epigenetic regulation of extracellular matrix homeostasis via Yap and Taz

Cells integrate mechanical cues to direct fate specification to maintain tissue function and homeostasis. While disruption of these cues is known to lead to aberrant cell behavior and chronic diseases, such as tendinopathies, the underlying mechanisms by which mechanical signals maintain cell function is not well understood. Here, we show using a novel model of tendon de-tensioning that loss of tensile cues in vivo acutely changes nuclear morphology, positioning, and expression of catabolic gene programs. Using paired ATAC/RNAseq, we further identify that a loss of cellular tension rapidly reduces chromatin accessibility in the vicinity of Yap/Taz genomic targets while also increasing expression of genes involved in matrix catabolism. Overexpression of Yap results in a reduction of chromatin accessibility at matrix catabolic gene loci, while also reducing transcriptional levels. Concordantly, depletion of Yap/Taz elevates matrix catabolic expression. Finally, we demonstrate that overexpression of Yap not only prevents the induction of a broad catabolic program following a loss of cellular tension, but also preserves the underlying chromatin state from force-induced alterations. Taken together, these results provide novel mechanistic details by which mechanical signals regulate tendon cell function to preserve matrix homeostasis through a Yap/Taz axis. Significance StatementCells integrate mechanical signals to regulate biological outputs within tissues. These processes are required for tissue function and homeostasis. Here, we show how mechanical cues (e.g. tension) directs tendon cell function and fate at a transcriptional and epigenetic level. Furthermore, we show that disruption of these mechanical cues leads to a disease-like cell state, indicating these mechanosensitive pathways could be important for diseases driven by perturbed mechanical signaling, such as tendinopathy. Finally, we demonstrate that genetic perturbation of a single protein can preserve cell and chromatin state following a loss of tension, supporting novel avenues for the development of innovative mechano-therapeutics.

cell biology↗

bioRxiv2021

Fabrication and delivery of mechano-activedmicrocapsules containing osteogenic factors in alarge animal model of osteochondral injury

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 poly(D,L-lactide-co-glycolide) (PLGA) microcapsules containing the pro-osteogenic agents triiodothyronine and {beta}-glycerophosphate, and delivered these microcapsules in a large animal model of osteochondral injury to preserve bone structure. We demonstrate that developed microcapsules ruptured in vitro under increasing mechanical loads, and readily sink within a liquid solution, allowing for gravity-based positioning onto the osteochondral surface. In a large animal, these mechano-active 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 time in 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 locally administered, 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 1 and 2 weeks after injection and surgery. Analysis of injected MAMCs showed that bioactive cargo was released in a controlled fashion over 2 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 establishes a new micro-fluidically generated delivery platform that releases therapeutic factors in an articulating joint, and reduces this to practice in the delivery of therapeutics that preserve bone structure after osteochondral injury.

bioengineering↗

bioRxiv2021

Marked differences in local bone remodeling in response to different marrow stimulation techniques in a large animal

Marrow stimulation, including subchondral drilling and microfracture, is the most commonly performed cartilage repair strategy, whereby the subchondral bone plate is perforated to release marrow-derived cells into a cartilage defect to initiate repair. Novel scaffolds and therapeutics are being designed to enhance and extend the positive short-term outcomes of this marrow stimulation. However, the translation of these newer treatments is hindered by bony abnormalities, including bone resorption, intralesional osteophytes, and bone cysts, that can arise after marrow stimulation. In this study, three different marrow stimulation approaches -- microfracture, subchondral drilling, and needle-puncture - were evaluated in a translationally relevant large animal model, the Yucatan minipig. The objective of this study was to determine which method of marrow access (malleted awl, drilled Kirschner wire, or spring-loaded needle) best preserved the underlying subchondral bone. Fluorochrome labels were injected at the time of surgery and 2 weeks post-surgery to capture bone remodeling over the first 4 weeks. Comprehensive outcome measures included cartilage indentation testing, histological grading, microcomputed tomography, and fluorochrome imaging. Our findings indicated that needle-puncture devices best preserved the underlying subchondral bone relative to other marrow access approaches. This may relate to the degree of bony compaction occurring with marrow access, as the Kirschner wire approach, which consolidated bone most, induced the most significant bone damage with marrow stimulation. This study provides basic science evidence in support of updated marrow stimulation techniques for preclinical and clinical practice.

bioengineering↗

bioRxiv2021

Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments

The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 (Fgf9), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. Taken together, we discovered that Fgf9 may play an influential role in muscle-bone crosstalk during embryonic and postnatal development.

developmental biology↗

bioRxiv2020

Success Criteria for Preclinical Testing of Cell-Instructive Hydrogels for Tendon Regeneration

Tendon injuries are difficult to heal in part because intrinsic tendon healing, which is dominated by scar tissue formation, does not effectively regenerate the native structure and function of healthy tendon. Further, many current treatment strategies also fall short of producing regenerated tendon with the native properties of healthy tendon. There is increasing interest in the use of cell-instructive strategies to limit the intrinsic fibrotic response following injury and improve the regenerative capacity of tendon in vivo. We have established multi-functional, cell-instructive hydrogels for treating injured tendon that afford tunable control over the biomechanical, biochemical, and structural properties of the cell microenvironments. Specifically, we incorporated integrin-binding domains (RGDS) and assembled multi-functional collagen mimetic peptides (mfCMPs) that enable cell adhesion and elongation of stem cells within synthetic hydrogels of designed biomechanical properties and evaluated these materials using targeted success criteria developed for testing in mechanically-demanding environments like tendon healing. The in vitro and in situ success criteria were determined based on systematic reviews of the most commonly reported outcome measures of hydrogels for tendon repair and established standards for testing of biomaterials. We then showed, using validation experiments, that multi-functional and synthetic hydrogels meet these criteria. Specifically, these hydrogels have mechanical properties comparable to developing tendon; are non-cytotoxic both in 2D bolus exposure (hydrogel components) and 3D encapsulation (full hydrogel); are formed, retained, and visualized within tendon defects over time (two-weeks); and provide mechanical support to tendon defects at the time of injection and in situ formation. Ultimately, the in vitro and in situ success criteria evaluated in this study were designed for preclinical research to rigorously test the potential to achieve successful tendon repair prior to in vivo testing and indicate the promise of multi-functional and synthetic hydrogels for continued translation. IMPACT STATEMENTTendon healing results in a weak scar that forms due to poor cell-mediated repair of the injured tissue. Treatments that tailor the instructions experienced by cells during healing afford opportunities to regenerate the healthy tendon. Engineered cell-instructive cues, including the biomechanical, biochemical, and structural properties of the cell microenvironment, within multi-functional synthetic hydrogels are promising therapeutic strategies for tissue regeneration. In this paper, the preclinical efficacy of multi-functional synthetic hydrogels for tendon repair is tested against rigorous in vitro and in situ success criteria. This study indicates the promise for continued preclinical translation of synthetic hydrogels for tissue regeneration.

bioengineering↗

bioRxiv2020

Mechanics and Differential Healing Outcomes of Small and Large Partial-Thickness Injuries of the Rat Rotator Cuff

The size of rotator cuff tears affects clinical outcomes following rotator cuff repair and is correlated with risk of re-injury. This study aimed to understand how defect size influences the structural and mechanical outcomes of the injured rotator cuff attachment in vivo. We used our previously established model of full-thickness injury of the rotator cuff tendon-bone attachment in Long Evans rats to compare differences in healing outcomes between small and large defects. Biomechanical properties, gross morphology, bone remodeling, and cell and tissue morphology were assessed at 3- or 8-weeks of healing. At the time of injury (no healing), large defects had decreased mechanical properties compared to small defects, and both defect sizes had decreased mechanical properties compared to intact attachments. The mechanical properties of the defect groups were comparable after 8-weeks of healing and significantly improved compared to no healing but failed to return to intact levels. Local bone volume at the defect site was higher in large compared to small defects on average and increased from 3- to 8-weeks. Contrastingly, bone quality, measured as bone volume percentage and trabecular morphometry, of the total epiphysis and greater tubercle decreased from 3- to 8-weeks of healing and these changes were not dependent on defect size. Qualitatively, we observed that large defects had increased disorganized collagen and neovascularization compared to small defects. In this study, we demonstrated that not only small but also large defects do not regenerate the mechanical and structural integrity of the intact rat rotator cuff attachment following healing in vivo. Statement of Clinical SignificanceOur rat model of full-thickness rotator cuff tears may be beneficial to understand and prevent tear enlargement in vivo.

bioengineering↗

bioRxiv2019

Photobiomodulation Does Not Influence Maturation and Leads to Mild Improvements in Functional Healing of Mouse Achilles Tendons

Tendon rupture can occur at any age and is commonly treated non-operatively, yet can result in persisting symptoms. Thus, a need exists to improve non-operative treatments of injured tendons. Photobiomodulation (PBM) therapy has shown promise in the clinic and is hypothesized to stimulate mitochondrial-related metabolism and improve healing. However, the effect of PBM therapy on mitochondrial function during tendon maturation and healing are unknown, and its effect on tendon structure and function remain unclear. In this study, near-infrared light (980:810nm blend, 2.5J/cm2) was applied at low (30mW/cm2) or high (300mW/cm2) irradiance to unilateral Achilles tendons of CD-1 mice during postnatal growth (maturation) as well as adult mice with bilateral Achilles tenotomy (healing). The chronic effect of PBM therapy on tendon structure and function was determined using histology and mechanics, and the acute effect of PBM therapy on mitochondrial-related gene expression was assessed. During maturation and healing, collagen alignment, cell number, and nuclear shape were unaffected by chronic PBM therapy. We found a sex-dependent effect of PBM therapy during healing on mechanical outcomes (e.g., increased stiffness and Youngs modulus for PBM-treated females, and increased strain at ultimate stress for PBM-treated males). Mitochondria-related gene expression was marginally influenced by PBM therapy for both maturation and healing studies. This study was the first to implement PBM therapy during both growth and healing of the murine tendon. PBM therapy resulted in marginal and sex-dependent effects on murine tendon.

bioengineering↗

bioRxiv2018

Partial-Width Injuries of the Rat Rotator Cuff Heal with Fibrosis

PurposeThe purpose of this study was to identify the healing outcomes following a partial-width, full-thickness injury to the rotator cuff tendon-bone attachment and establish if the adult attachment can regenerate the morphology of the healthy attachment.\n\nHypothesisWe hypothesized that a partial-width injury to the attachment would heal via fibrosis and bone remodeling, resulting in increased cellularity and extracellular matrix deposition, reduced bone volume, osteoclast presence and decreased collagen organization compared to shams.\n\nMaterials and MethodsA biopsy punch was used to create a partial-width injury at the center one-third of the rat infraspinatus attachment, and the contralateral limb underwent a sham operation. Rats were sacrificed at 3- and 8-weeks after injury for analyses. Analyses performed at each time-point included cellularity (Hematoxylin & Eosin), ECM deposition (Massons Trichrome), bone volume (micro-computed tomography; microCT), osteoclast activity (Tartrate Resistant Acid Phosphatase; TRAP), and collagen fibril organization (Picrosirius Red). Injured and sham shoulders were compared at both 3- and 8-weeks using paired, two-way ANOVAs with repeated measures and Sidaks correction for multiple comparisons.\n\nResultsCellularity and ECM deposition increased at both 3- and 8-weeks compared to sham contralateral attachments. Bone volume decreased and osteoclast presence increased at both 3- and 8-weeks compared to sham contralateral limbs. Collagen fibril organization was reduced at 3-weeks after injury compared to 3-week sham attachments.\n\nConclusionsThese findings suggest that a partial-width injury to the rotator cuff attachment does not fully regenerate the native structure of the healthy attachment. The injury model healed via scar-like fibrosis and did not propagate into a full-width tear after 8-weeks of healing.

bioengineering↗

bioRxiv2018

A novel model for the induction of postnatal murine hip deformity

Acetabular dysplasia is a recognized cause of hip osteoarthritis (OA). A paucity of animal models exists to investigate structural and functional changes that mediate morphology of the dysplastic hip and drive the subsequent arthritic cascade. Utilizing a novel murine model, this study investigated the role of surgically-induced unilateral instability of the postnatal hip on the initiation and progression of acetabular dysplasia and impingement up to 8-weeks post-injury. Specifically, C57BL6 mice were used to develop titrated levels of hip instability (mild, moderate, severe, and femoral head removal) at 3-weeks of age, a critical time for hip maturation. Joint shape, acetabular coverage, histomorphology, immunohistochemistry, and statistical shape modeling were used to assess overall quality of joint health and three-dimensional hip shape following 8 weeks of titrated destabilization. This titrated approach included mild, moderate, severe, and complete instability via surgical destabilization of the murine hip. Acetabular coverage was reduced following severe, but not moderate, instability. Moderate instability induced lateralization of the femoral head without dislocation, whereas severe instability led to complete dislocation and formation of pseudoacetabula. Mild instability did not result in statistically significant morphological changes to the hip. Complete destabilization via femoral head removal led to reduced joint space volume and reduced bone volume ratio in the remnant proximal femur. Collectively, these results support the notion that hip instability, driven by loss of function, leads to morphometric changes in the maturing mouse hip. This model could be useful for future studies investigating the mechanical and cellular adaptations to hip instability during maturation.

pathology↗