Tissue engineered vascular grafts transform into autologous neovessels capable of native function and growth.

Background: Tissue-engineered vascular grafts (TEVGs) have the potential to advance the surgical management of infants and children requiring congenital heart surgery by creating functional vascular conduits with growth capacity. Methods: Herein, we used an integrative computational-experimental approach to elucidate the natural history of neovessel formation in a large animal preclinical model; combining an in vitro accelerated degradation study with mechanical testing, large animal implantation studies with in vivo imaging and histology, and data-informed computational growth and remodeling models. Results: Our findings demonstrate that the structural integrity of the polymeric scaffold is lost over the first 26 weeks in vivo, while polymeric fragments persist for up to 52 weeks. Our models predict that early neotissue accumulation is driven primarily by inflammatory processes in response to the implanted polymeric scaffold, but that turnover becomes progressively mechano-mediated as the scaffold degrades. Using a lamb model, we confirm that early neotissue formation results primarily from the foreign body reaction induced by the scaffold, resulting in an early period of dynamic remodeling characterized by transient TEVG narrowing. As the scaffold degrades, mechano-mediated neotissue remodeling becomes dominant around 26 weeks. After the scaffold degrades completely, the resulting neovessel undergoes growth and remodeling that mimicks native vessel behavior, including biological growth capacity, further supported by fluid-structure interaction simulations providing detailed hemodynamic and wall stress information. Conclusions: These findings provide insights into TEVG remodeling, and have important implications for clinical use and future development of TEVGs for children with congenital heart disease.

Predictors of poor pre-operative psychological status among patients with cartilage defects.

BACKGROUND: We evaluated the risk factors for pain catastrophizing, kinesiophobia, and elevated depressive symptoms among patients undergoing high-grade cartilage defect surgery. We hypothesized that cartilage patients would demonstrate high scores on pain catastrophizing, kinesiophobia, and depression testing prior to surgery. METHODS: Two hundred and ten patients undergoing surgery for high-grade cartilage defects (56% chondroplasty, 36% microfracture, 22% autologous chondrocyte implantation) completed a preoperative survey before undergoing surgery. Outcome scores assessed were: International Knee Documentation Committee-Symptom (IKDC-S) score, Tegner activity score, Pain catastrophizing scale (PCS), Tampa scale for kinesiophobia (TSK-11), and Patient Health Questionnaire depression scale (PHQ-9). Multivariate logistic regression was used to determine what pre-operative factors predicted pain catastrophizing, kinesiophobia, and elevated depressive symptoms. RESULTS: The mean pre-operative Tegner score was 5.8 (SD 2.4) and IKDC-S score was 44.7 (SD 11.1). Prior to surgery, 19% had abnormal pain catastrophizing (PCS ≥ 20 points), 14.4% had moderate-severe depression (PHQ ≥ 10), and 49.0% had high kinesiophobia (TSK-11 ≥ 25). Lower pre-operative Tegner scores predicted moderate-severe depressive symptoms (per point decrease, OR 1.36, 95% CI 1.06, 1.76; p = 0.008). Predictors of elevated pain catastrophizing were lower pre-operative IKDC-S scores (per 5-point decrease, OR 1.28, 95% CI 1.08, 1.51; p = 0.002) and symptom duration >6 months (OR 2.20 CI 1.14, 4.32; p = 0.02). A lower pre-operative IKDC-S score (per 5-point decrease, OR 1.17, CI 1.03, 1.33; p = 0.02) predicted elevated kinesiophobia. CONCLUSION: Low self-reported function, low activity level and symptom duration greater than six months are associated with poor preoperative psychological status.

The Real Need for Regenerative Medicine in the Future of Congenital Heart Disease Treatment.

Bioabsorbable materials made from polymeric compounds have been used in many fields of regenerative medicine to promote tissue regeneration. These materials replace autologous tissue and, due to their growth potential, make excellent substitutes for cardiovascular applications in the treatment of congenital heart disease. However, there remains a sizable gap between their theoretical advantages and actual clinical application within pediatric cardiovascular surgery. This review will focus on four areas of regenerative medicine in which bioabsorbable materials have the potential to alleviate the burden where current treatment options have been unable to within the field of pediatric cardiovascular surgery. These four areas include tissue-engineered pulmonary valves, tissue-engineered patches, regenerative medicine options for treatment of pulmonary vein stenosis and tissue-engineered vascular grafts. We will discuss the research and development of biocompatible materials reported to date, the evaluation of materials in vitro, and the results of studies that have progressed to clinical trials.

Deconstructing tissue engineered trachea: Assessing the role of synthetic scaffolds, segmental replacement and cell seeding on graft performance.

The ideal construct for tracheal replacement remains elusive in the management of long segment airway defects. Tissue engineered tracheal grafts (TETG) have been limited by the development of graft stenosis or collapse, infection, or lack of an epithelial lining. We applied a mouse model of orthotopic airway surgery to assess the impact of three critical barriers encountered in clinical applications: the scaffold, the extent of intervention, and the impact of cell seeding and characterized their impact on graft performance. First, synthetic tracheal scaffolds electrospun from polyethylene terephthalate / polyurethane (PET/PU) were orthotopically implanted in anterior tracheal defects of C57BL/6 mice. Scaffolds demonstrated complete coverage with ciliated respiratory epithelium by 2 weeks. Epithelial migration was accompanied by macrophage infiltration which persisted at long term (>6 weeks) time points. We then assessed the impact of segmental tracheal implantation using syngeneic trachea as a surrogate for the ideal tracheal replacement. Graft recovery involved local upregulation of epithelial progenitor populations and there was no evidence of graft stenosis or necrosis. Implantation of electrospun synthetic tracheal scaffold for segmental replacement resulted in respiratory distress and required euthanasia at an early time point. There was limited epithelial coverage of the scaffold with and without seeded bone marrow-derived mononuclear cells (BM-MNCs). We conclude that synthetic scaffolds support re-epithelialization in orthotopic patch implantation, syngeneic graft integration occurs with focal repair mechanisms, however epithelialization in segmental synthetic scaffolds is limited and is not influenced by cell seeding. STATEMENT OF SIGNIFICANCE: The life-threatening nature of long-segment tracheal defects has led to clinical use of tissue engineered tracheal grafts in the last decade for cases of compassionate use. However, the ideal tracheal reconstruction using tissue-engineered tracheal grafts (TETG) has not been clarified. We addressed the core challenges in tissue engineered tracheal replacement (re-epithelialization and graft patency) by defining the role of cell seeding with autologous bone marrow-derived mononuclear cells, the mechanism of respiratory epithelialization and proliferation, and the role of the inflammatory immune response in regeneration. This research will facilitate comprehensive understanding of cellular regeneration and neotissue formation on TETG, which will permit targeted therapies for accelerating re-epithelialization and attenuating stenosis in tissue engineered airway replacement.

Mast Cell/Proteinase Activated Receptor 2 (PAR2) Mediated Interactions in the Pathogenesis of Discogenic Back Pain.

Mast cells (MCs) are present in the painful degenerate human intervertebral disc (IVD) and are associated with disease pathogenesis. MCs release granules containing enzymatic and inflammatory factors in response to stimulants or allergens. The serine protease, tryptase, is unique to MCs and its activation of the G-protein coupled receptor, Protease Activated Receptor 2 (PAR2), induces inflammation and degradation in osteoarthritic cartilage. Our previously published work has demonstrated increased levels of MC marker tryptase in IVD samples from discogenic back pain patients compared to healthy control IVD samples including expression of chemotactic agents that may facilitate MC migration into the IVD. To further elucidate MCs' role in the IVD and mechanisms underlying its effects, we investigated whether (1) human IVD cells can promote MC migration, (2) MC tryptase can mediate up-regulation of inflammatory/catabolic process in human IVD cells and tissue, and (3) the potential of PAR2 antagonist to function as a therapeutic drug in in vitro human and ex vivo bovine pilot models of disease. MC migration was quantitatively assessed using conditioned media from primary human IVD cells and MC migration examined through Matrigel. Exposure to soluble IVD factors significantly enhanced MC migration, suggesting IVD cells can recruit MCs. We also demonstrated significant upregulation of MC chemokine SCF and angiogenic factor VEGFA gene expression in human IVD cells in vitro in response to recombinant human tryptase, suggesting tryptase can enhance recruitment of MCs and promotion of angiogenesis into the usually avascular IVD. Furthermore, tryptase can degrade proteoglycans in IVD tissue as demonstrated by significant increases in glycosaminoglycans released into surrounding media. This can create a catabolic microenvironment compromising structural integrity and facilitating vascular migration usually inhibited by the anti-angiogenic IVD matrix. Finally, as a "proof of concept" study, we examined the therapeutic potential of PAR2 antagonist (PAR2A) on human IVD cells and bovine organ culture IVD model. While preliminary data shows promise and points toward structural restoration of the bovine IVD including down-regulation of VEGFA, effects of PAR2 antagonist on human IVD cells differ between gender and donors suggesting that further validation is required with larger cohorts of human specimens.

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model.

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

The chondrodystrophic dog: A clinically relevant intermediate-sized animal model for the study of intervertebral disc-associated spinal pain.

Low back pain (LBP) is the leading cause of disability worldwide, with an estimated 80% of the American population suffering from a painful back condition at some point during their lives. The most common cause of LBP is intervertebral disc (IVD) degeneration (IVDD), a condition that can be difficult to treat, either surgically or medically, with current available therapies. Thus, understanding the pathological mechanisms of IVDD and developing novel treatments are critical for improving outcome and quality of life in people living with LBP. While experimental animal models provide valuable mechanistic insight, each model has limitations that complicate translation to the clinical setting. This review focuses on the chondrodystrophic canine clinical model of IVDD as a promising model to assess IVD-associated spinal pain and translational therapeutic strategies for LBP. The canine IVD, while smaller in size than human, goat, ovine, and bovine IVDs, is larger than most other small animal IVDD models and undergoes maturational changes similar to those of the human IVD. Furthermore, both dogs and humans develop painful IVDD as a spontaneous process, resulting in similar characteristic pathologies and clinical signs. Future exploration of the canine model as a model of IVD-associated spinal pain and biological treatments using the canine clinical model will further demonstrate its translational capabilities with the added ethical benefit of treating an existing veterinary patient population with IVDD.

Deconstructing the Tissue Engineered Vascular Graft: Evaluating Scaffold Pre-Wetting, Conditioned Media Incubation, and Determining the Optimal Mononuclear Cell Source.

Stenosis limits widespread use of tissue-engineered vascular grafts (TEVGs), and bone marrow mononuclear cell (BM-MNC) seeding attenuates this complication. Yet seeding is a multistep process, and the singular effects of each component are unknown. We investigated which components of the clinical seeding protocol confer graft patency and sought to identify the optimal MNC source. Scaffolds composed of polyglycolic acid and ε-caprolactone/ι-lactic acid underwent conditioned media (CM) incubation (n = 25) and syngeneic BM-MNC (n = 9) or peripheral blood (PB)-MNC (n = 20) seeding. TEVGs were implanted for 2 weeks in the mouse IVC. CM incubation and PB-MNC seeding did not increase graft patency compared to control scaffolds prewet with PBS (n = 10), while BM-MNC seeding reduced stenosis by suppressing inflammation and smooth muscle cell, myofibroblast, and pericyte proliferation. IL-1ß, IL-6, and TNFα were elevated in the seeded BM-MNC supernatant. Further, BM-MNC seeding reduced platelet activation in a dose-dependent manner, possibly contributing to TEVG patency.

Mast Cell-Intervertebral disc cell interactions regulate inflammation, catabolism and angiogenesis in Discogenic Back Pain.

Low back pain (LBP) is a widespread debilitating disorder of significant socio-economic importance and intervertebral disc (IVD) degeneration has been implicated in its pathogenesis. Despite its high prevalence the underlying causes of LBP and IVD degeneration are not well understood. Recent work in musculoskeletal degenerative diseases such as osteoarthritis have revealed a critical role for immune cells, specifically mast cells in their pathophysiology, eluding to a potential role for these cells in the pathogenesis of IVD degeneration. This study sought to characterize the presence and role of mast cells within the IVD, specifically, mast cell-IVD cell interactions using immunohistochemistry and 3D in-vitro cell culture methods. Mast cells were upregulated in painful human IVD tissue and induced an inflammatory, catabolic and pro-angiogenic phenotype in bovine nucleus pulposus and cartilage endplate cells at the gene level. Healthy bovine annulus fibrosus cells, however, demonstrated a protective role against key inflammatory (IL-1ß and TNFα) and pro-angiogenic (VEGFA) genes expressed by mast cells, and mitigated neo-angiogenesis formation in vitro. In conclusion, mast cells can infiltrate and elicit a degenerate phenotype in IVD cells, enhancing key disease processes that characterize the degenerate IVD, making them a potential therapeutic target for LBP.

The role of myeloid cell-derived PDGF-B in neotissue formation in a tissue-engineered vascular graft.

AIM: Inflammatory myeloid lineage cells mediate neotissue formation in tissue-engineered vascular grafts, but the molecular mechanism is not completely understood. We examined the role of vasculogenic PDGF-B in tissue-engineered vascular graft neotissue development. MATERIALS & METHODS: Myeloid cell-specific PDGF-B knockout mice (PDGF-KO) were generated using bone marrow transplantation, and scaffolds were implanted as inferior vena cava interposition grafts in either PDGF-KO or wild-type mice. RESULTS: After 2 weeks, grafts from PDGF-KO mice had more remaining scaffold polymer and less intimal neotissue development. Increased macrophage apoptosis, decreased smooth muscle cell proliferation and decreased collagen content was also observed. CONCLUSION: Myeloid cell-derived PDGF contributes to vascular neotissue formation by regulating macrophage apoptosis, smooth muscle cell proliferation and extracellular matrix deposition.

The Contribution of Biomechanical-Biological Interactions of the Spine to Low Back Pain.

OBJECTIVE: The objective of this mini-review is to examine a subset of literature that demonstrates multiple interactions between mechanics and biology within the spine and propose how incorporation of these mechano-biologic interactions can be applied to improve the conceptual understanding of tissue tolerances. BACKGROUND: Low back pain represents a major musculoskeletal problem in the workplace. Traditional biomechanical assessments have employed tissue tolerances as an approach for reducing workplace injuries; however, development of more universal biologically sensitive tolerances requires incorporation of mechano-biologic interactions. METHODS: A focused literature review addressing the interactions between mechanical loading and biology in the spine. RESULTS: Mechanical loads applied to the body are distributed across all spatial scales from the body to the tissues to the cells. These mechanical loads regulate cellular metabolism and over time can lead to tissue strengthening or weakening. Mechanical loading also interacts with the biologic environment (e.g., tissue inflammation, nerve sensitization) to influence the perception of pain, thereby changing the risk of experiencing pain. Biologic tissues also exhibit time-dependent changes in mechanical behaviors that occur throughout the day and with disease, suggesting tissue tolerances are time dependent. CONCLUSION: Incorporating mechano-biologic interactions into the traditional tissue tolerance paradigm through describing tissue tolerances as a function of multiple factors (e.g., preexisting risk factors, underlying pathology, and time) may lead to the development of tissue tolerances that are more representative of the in vivo situation. APPLICATION: Efforts must work toward incorporating biological concepts into tissue tolerances in order to improve risk assessment tools.