Porous honeycomb film membranes enhance endothelial barrier integrity in human vascular wall bilayer model compared to standard track-etched membranes.

In vitro vascular wall bilayer models for drug testing and disease modeling must emulate the physical and biological properties of healthy vascular tissue and its endothelial barrier function. Both endothelial cell (EC)-vascular smooth muscle cell (SMC) interaction across the internal elastic lamina (IEL) and blood vessel stiffness impact endothelial barrier integrity. Polymeric porous track-etched membranes (TEM) typically represent the IEL in laboratory vascular bilayer models. However, TEM stiffness exceeds that of diseased blood vessels, and the membrane pore architecture limits EC-SMC interaction. The mechanical properties of compliant honeycomb film (HCF) membranes better simulate the Young's modulus of healthy blood vessels, and HCFs are thinner (4 vs. 10 µm) and more porous (57 vs. 6.5%) than TEMs. We compared endothelial barrier integrity in vascular wall bilayer models with human ECs and SMCs statically cultured on opposite sides of HCFs and TEMs (5 µm pores) for up to 12 days. Highly segregated localization of tight junction (ZO-1) and adherens junction (VE-cadherin) proteins and quiescent F-actin cytoskeletons demonstrated superior and earlier maturation of interendothelial junctions. Quantifying barrier integrity based on transendothelial electrical resistance (TEER), membranes showed only minor but significant TEER differences despite enhanced junctional protein localization on HCF. Elongated ECs on HCF likely experienced greater paracellular diffusion than blocky ECs on TEM. Also, larger populations of plaques of connexin 43 subunit-containing gap junctions suggested enhanced EC-SMC communication across the more porous, thinner HCF. Compared with standard TEMs, engineered vascular wall bilayers cultured on HCFs better replicate physiologic endothelial barrier integrity.

Human Vascular Wall Microfluidic Model for Preclinical Evaluation of Drug-Induced Vascular Injury.

Drug-induced vascular injury (DIVI) in preclinical animal models often leads to candidate compound termination during drug development. DIVI has not been documented in human clinical trials with drugs that cause DIVI in preclinical animals. A robust human preclinical assay for DIVI is needed as an early vascular injury screen. A human vascular wall microfluidic tissue chip was developed with a human umbilical vein endothelial cell (HUVEC)-umbilical artery smooth muscle cell (vascular smooth muscle cell, VSMC) bilayer matured under physiological shear stress. Optimized temporal flow profiles produced HUVEC-VSMC bilayers with quiescent endothelial cell (EC) monolayers, EC tight junctions, and contractile VSMC morphology. Dose-response testing (3-30 µM concentration) was conducted with minoxidil and tadalafil vasodilators. Both drugs have demonstrated preclinical DIVI but lack clinical evidence. The permeability of severely damaged engineered bilayers (30 µM tadalafil) was 4.1 times that of the untreated controls. Immunohistochemical protein assays revealed contrasting perspectives on tadalafil and minoxidil-induced damage. Tadalafil impacted the endothelial monolayer with minor injury to the contractile VSMCs, whereas minoxidil demonstrated minor EC barrier injury but damaged VSMCs and activated ECs in a dose-response manner. This proof-of-concept human vascular wall bilayer model of DIVI is a critical step toward developing a preclinical human screening assay for drug development. Impact statement More than 90% of drug candidates fail during clinical trials due to human efficacy and toxicity concerns. Preclinical studies rely heavily on animal models, although animal toxicity and drug metabolism responses often differ from humans. During the drug development process, perfused in vitro human tissue chips could model the clinical drug response and potential toxicity of candidate compounds. Our long-term objective is to develop a human vascular wall tissue chip to screen for drug-induced vascular injury. Its application could ultimately reduce drug development delays and costs, and improve patient safety.

Single-walled carbon nanohorns modulate tenocyte cellular response and tendon biomechanics.

Subfailure ligament and tendon injury remain a significant burden to global healthcare. Here, we present the use of biocompatible single-walled carbon nanohorns (CNH) as a potential treatment for the repair of sub-failure injury in tendons. First, in vitro exposure of CNH to human tenocytes revealed no change in collagen deposition but a significant decrease in cell metabolic activity after 14 days. Additionally, gene expression studies revealed significant downregulation of collagen Types I and III mRNA at 7 days with some recovery after 14 days of exposure. Biomechanical tests with explanted porcine digitorum tendons showed the ability of CNH suspensions to modulate tendon biomechanics, most notably elastic moduli immediately after treatment. in vivo experiments demonstrated the ability of CNH to persist in the damaged matrix of stretch-injured Sprague Dawley rat Achilles tendon but not significantly modify tendon biomechanics after 7 days of treatment. Although these results demonstrate the early feasibility of utility of CNH as a potential modality for tendon subfailure injury, additional work is needed to further validate and ensure clinical efficacy.

In vivo response to decellularized mesothelium scaffolds.

Biological surgical scaffolds are used in plastic and reconstructive surgery to support structural reinforcement and regeneration of soft tissue defects. Macrophage and fibroblast cell populations heavily regulate scaffold integration into host tissue following implantation. In the present study, the biological host response to a commercially available surgical scaffold (Meso BioMatrix Surgical Mesh (MBM)) was investigated for up to 9 weeks after subcutaneous implantation; this scaffold promoted superior cell migration and infiltration previously in in vitro studies relative to other commercially available scaffolds. Infiltrating macrophages and fibroblasts phenotypes were assessed for evidence of inflammation and remodeling. At week 1, macrophages were the dominant cell population, but fibroblasts were most abundant at subsequent time points. At week 4, the scaffold supported inflammation modulation as indicated by M1 to M2 macrophage polarization; the foreign body giant cell response resolved by week 9. Unexpectedly, a fibroblast subpopulation expressed macrophage phenotypic markers, following a similar trend in transitioning from a proinflammatory to anti-inflammatory phenotype. Also, α-smooth muscle actin-expressing myofibroblasts were abundant at weeks 4 and 9, mirroring collagen expression and remodeling activity. MBM supported physiologic responses observed during normal wound healing, including cellular infiltration, host tissue ingrowth, remodeling of matrix proteins, and immune modulation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 716-725, 2018.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro.

BACKGROUND: Proliferative therapy, or prolotherapy, is a controversial treatment method for many connective tissue injuries and disorders. It involves the injection of a proliferant, or irritant solution, into the site of injury, which causes small-scale cell death. This therapeutic trauma is theorized to initiate the body's wound-healing cascade, perhaps leading to tissue repair. The immediate effects of many of these proliferants are poorly characterized, as are the cellular responses to them; here, we sought to evaluate the immediate effects of two common proliferants (dextrose and P2G, a combination of phenol, glucose, and glycerin) on the cellular response of human tenocytes, and begin to explicate the mechanisms with which each proliferant functions. QUESTIONS/PURPOSES: We asked: What are the effects of treating cultured tenocytes with proliferative treatment agents on their (1) cellular metabolic activity, (2) RNA expression, (3) protein secretion, and (4) cell migration? METHODS: Using human hamstring and Achilles tendon cells, we attempted to answer our research questions. We used a colorimetric metabolic assay to assess the effect of dextrose and P2G proliferant treatment on cell mitochondrial activity compared with nontreated tenocytes. Next, using quantitative PCR, ELISA, and a reporter cell line, we assessed the expression of several key markers involved in tendon development and inflammation. In addition, we used a scratch wound-healing assay to evaluate the effect of proliferant treatment on tenocyte migration. RESULTS: Results showed that exposure to both solutions led to decreased metabolic activity of tenocytes, with P2G having the more pronounced effect (75% ± 7% versus 95% ± 7% of untreated control cell metabolic levels) (ANOVA; p < 0.01; mean difference, 0.202; 95% CI, 0.052-0.35). Next, gene expression analysis confirmed that treatment led to the upregulation of key proinflammatory markers including interleukin-8 and cyclooxygenase-2 and downregulation of the matrix marker collagen type I. Furthermore, using a reporter cell line for transforming growth factor-ß (TGF-ß), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-ß bioactivity. Analysis of soluble proteins using ELISA revealed elevated levels of soluble prostaglandin E2 (PGE2), a prominent inducer of inflammation. Finally, both solutions led to decreased cellular migration in the tenocytes. CONCLUSIONS: Taken together, these results suggest that prolotherapy, more so with P2G, may work by decreasing cellular function and eliciting an inflammatory response in tenocytes. Additional studies are needed to confirm the cellular signaling mechanisms involved and the resulting immediate response in vivo. CLINICAL RELEVANCE: If these preliminary in vitro findings can be confirmed in an in vivo model, they may provide clues for a possible cellular mechanism of a common alternative treatment method currently used for certain soft tissue injuries.

Hypoxia Impairs Mesenchymal Stromal Cell-Induced Macrophage M1 to M2 Transition.

The transition of macrophages from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype is crucial for the progression of normal wound healing. Persistent M1 macrophages within the injury site may lead to an uncontrolled macrophage-mediated inflammatory response and ultimately a failure of the wound healing cascade, leading to chronic wounds. Mesenchymal stromal cells (MSCs) have been widely reported to promote M1 to M2 macrophage transition; however, it is unclear whether MSCs can drive this transition in the hypoxic environment typically observed in chronic wounds. Here we report on the effect of hypoxia (1% O2) on MSCs' ability to transition macrophages from the M1 to the M2 phenotype. While hypoxia had no effect on MSC secretion, it inhibited MSC-induced M1 to M2 macrophage transition, and suppressed macrophage expression and production of the anti-inflammatory mediator interleukin-10 (IL-10). These results suggest that hypoxic environments may impede the therapeutic effects of MSCs.

Cross-Talk Between Human Tenocytes and Bone Marrow Stromal Cells Potentiates Extracellular Matrix Remodeling In Vitro.

Tendon and ligament (T/L) pathologies account for a significant portion of musculoskeletal injuries and disorders. Tissue engineering has emerged as a promising solution in the regeneration of both tissues. Specifically, the use of multipotent human mesenchymal stromal cells (hMSC) has shown great promise to serve as both a suitable cell source for tenogenic regeneration and a source of trophic factors to induce tenogenesis. Using four donor sets, we investigated the bidirectional paracrine tenogenic response between human hamstring tenocytes (hHT) and bone marrow-derived hMSC. Cell metabolic assays showed that only one hHT donor experienced sustained notable increases in cell metabolic activity during co-culture. Histological staining confirmed that co-culture induced elevated collagen protein levels in both cell types at varying time-points in two of four donor sets assessed. Gene expression analysis using qPCR showed the varied up-regulation of anabolic and catabolic markers involved in extracellular matrix maintenance for hMSC and hHT. Furthermore, analysis of hMSC/hHT co-culture secretome using a reporter cell line for TGF-ß, a potent inducer of tenogenesis, revealed a trend of higher TGF-ß bioactivity in hMSC secretome compared to hHT. Finally, hHT cytoskeletal immunostaining confirmed that both cell types released soluble factors capable of inducing favorable tenogenic morphology, comparable to control levels of soluble TGF-ß1. These results suggest a potential for TGF-ß-mediated signaling mechanism that is involved during the paracrine interplay between the two cell types that is reminiscent of T/L matrix remodeling/turnover. These findings have significant implications in the clinical use of hMSC for common T/L pathologies.

High elastic modulus nanoparticles: a novel tool for subfailure connective tissue matrix damage.

Subfailure matrix injuries such as sprains and strains account for a considerable portion of ligament and tendon pathologies. In addition to the lack of a robust biological healing response, these types of injuries are often characterized by seriously diminished matrix biomechanics. Recent work has shown nanosized particles, such as nanocarbons and nanocellulose, to be effective in modulating cell and biological matrix responses for biomedical applications. In this article, we investigate the feasibility and effect of using high stiffness nanostructures of varying size and shape as nanofillers to mechanically reinforce damaged soft tissue matrices. To this end, nanoparticles (NPs) were characterized using atomic force microscopy and dynamic light scattering techniques. Next, we used a uniaxial tensile injury model to test connective tissue (porcine skin and tendon) biomechanical response to NP injections. After injection into damaged skin and tendon specimens, the NPs, more notably nanocarbons in skin, led to an increase in elastic moduli and yield strength. Furthermore, rat primary patella tendon fibroblast cell activity evaluated using the metabolic water soluble tetrazolium salt assay showed no cytotoxicity of the NPs studied, instead after 21 days nanocellulose-treated tenocytes exhibited significantly higher cell activity when compared with nontreated control tenocytes. Dispersion of nanocarbons injected by solution into tendon tissue was investigated through histologic studies, revealing effective dispersion and infiltration in the treated region. Such results suggest that these high modulus NPs could be used as a tool for damaged connective tissue repair.

Effect of prolotherapy on cellular proliferation and collagen deposition in MC3T3-E1 and patellar tendon fibroblast populations.

Proliferative therapy, or prolotherapy, is a treatment for damaged connective tissues involving the injection of a solution (proliferant) which causes local cell death and triggers the body's wound healing cascade. Physicians vary in their use of this technique; it is employed for ligaments but has also been investigated for tissues such as cartilage. Physicians also vary in treatment regiments using different dosses of the proliferant. This study evaluates several proliferant dosages develop an optimal dosage that maximizes cell and collagen regeneration. This study also looks at cell and collagen regeneration in response to proliferant exposure outside of the healing cascade. MC3T3-E1 cells and patellar tendon fibroblasts were exposed to various amounts of the proliferant P2G and monitored over several weeks. The results showed an inverse relationship between proliferant concentration and cell viability and collagen production in MC3T3-E1 cells. Following exposure, cell populations experienced an initial decrease in cell number followed by increased proliferation. Trichrome staining over 4 weeks showed an increase in collagen production after proliferant exposure. However the cell numbers and amounts of collagen from the treated groups never surpassed those of the untreated groups, although collagen production was comparable in fibroblasts. The results of this basic study show that there is an effective proliferant dosage and point to a local response to the proliferant that increases cell proliferation and collagen production separate from the wound healing cascade. This local response may not be adequate for complete healing and assistance from the body's healing cascade may be required.

Evaluation of a hydrogel-fiber composite for ACL tissue engineering.

The anterior cruciate ligament (ACL) is necessary for normal knee stability and movement. Unfortunately the ACL is also the most frequently injured ligament of the knee with severe disruptions requiring surgical intervention. In response to this, tissue engineering has emerged as an option for ACL replacement and repair. In this study we present a novel hydrogel-fibrous scaffold as a potential option for ACL replacement. The scaffold was composed of PLLA fibers, in a previously evaluated braid-twist structure, combined with a polyethylene glycol diacrylate (PEGDA) hydrogel to improve viscoelastic properties. Both hydrogel concentration (10%, 15%, and 20%) and amount of hydrogel (soaking the fibrous scaffold in hydrogel solution or encasing the scaffold in a block of hydrogel) were evaluated. It was found that the braid-twist scaffold had a greater porosity and larger number of pores above 100 µm than braided scaffolds with the same braiding angle. After testing for their effects on swelling, fiber degradation, and protein release, as well as viscoelastic and tensile testing (when combined with fibrous scaffolds), it was found that the composite scaffold soaked in 10% hydrogel had the best chemical release and mechanical properties. The optimized structure behaved similarly to natural ligament in tension with the addition of the hydrogel decreasing the ultimate tensile stress (UTS), but the UTS was still comparable to natural ACL. In addition, cellular studies showed that the hydrogel-PLLA fiber composite supported fibroblast growth.