Emmanuel C. Ekwueme

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.

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 bodys 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 bodys healing cascade may be required.

Novel matrix based anterior cruciate ligament (ACL) regeneration

Among the five ligaments in the knee the anterior cruciate ligament (ACL) is among the most important for stability and also the most commonly injured. Due to a lack of vascularization, the ACL has poor healing potential, therefore moderate to severe damage warrants medical intervention. Ligaments are complex, highly organized tissues; they are longitudinally arranged with a great deal of order that begins at the molecular level and carries through to the tissue level. The components of the ligament and their location and orientation heavily influence the tissues mechanical behavior. ACL replacements have faced a variety of limitations that prevented their extensive use, including implant fatigue or fraying of the device. In the face of these problems, investigators have begun to examine a variety of matrix based techniques to create options for ACL repair, replacement, and regeneration. This article will discuss ACL structure and mechanics, past replacement options and their limitations, and recent tissue engineered options for ACL repair. These techniques employ a wide variety of designs, materials, and methods to heal damaged ACL tissue or regenerate lost tissue in order to regain full ACL strength and mechanics.

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‐β, a potent inducer of tenogenesis, revealed a trend of higher TGF‐β 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‐β1. These results suggest a potential for TGF‐β‐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. J. Cell. Biochem. 117: 684–693, 2016.

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.

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.

Prolotherapy Induces an Inflammatory Response in Human Tenocytes In Vitro

BackgroundProliferative 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/purposesWe 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?MethodsUsing 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.ResultsResults 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-β (TGF-β), a prominent antiinflammatory marker, we showed that treatments led to decreased TGF-β 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.ConclusionsTaken 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 RelevanceIf 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.

Recent Advancements in Ligament Replacement

The anterior cruciate ligament (ACL) is important for knee stability and kinematics. It is also the most com- monly injured ligament of the knee and due to its poor healing potential, severe damage warrants surgical intervention in- cluding complete replacement. Therefore, investigators have begun to pursue new techniques and devices for the repair, regeneration, and replacement of the ACL. These options involve the use of mechanically functional grafts that are designed to increase implant stability in order to withstand normal mechanical loads (while promoting ligament develop- ment in some cases). This article presents background on the ACL and its replacement, novel replacement approaches utilizing a variety of materials, and recent patent coverage.

Nanostructure-enhanced proliferative therapy for ligaments and tendons

Due to their lack of sufficient vascularization, ligament and tendon healing can be lengthy and cumbersome. Proliferative therapy, or prolotherapy, is a somewhat controversial alternative treatment for these injuries that involves the injection of a solution that causes initial small-scale cell damage to induce a robust injury response for tissue healing. The combination of proliferative therapy with carbon nanostructures to produce a living composite ligament may significantly improve healing and stability of damaged ligaments and tendons. While the prolotherapy would induce a healing response that causes the fibroblasts in the tissue to produce new collagen, the addition of the nanostructures could immediately improve strength; preventing further injury and further strengthening the tissue once it has been healed by serving as a constant irritant to fibroblasts to produce structural ECM. In several in vitro studies, the cellular viability of rat patellar tendon fibroblasts in response to the different treatments was measured. After treatment, cell populations experienced increases in cellular viability. The results of these basic studies show the potential of prolotherapy and carbon nanostructures in ligament and tendon healing at a fundamental, cellular level. They also point to another mechanism for tissue healing after prolotherapy that is separate from the bodys wound healing cascade.

Nanostructure-Enhanced Proliferative Therapy for Ligaments and Tendons

Due to their lack of sufficient vascularization, ligament and tendon healing can be lengthy and cumbersome. Proliferative therapy, or prolotherapy, is a somewhat controversial alternative treatment for these injuries that involves the injection of a solution that causes initial small-scale cell damage to induce a robust injury response for tissue healing. The combination of proliferative therapy with carbon nanostructures to produce a living composite ligament may significantly improve healing and stability of damaged ligaments and tendons. While the prolotherapy would induce a healing response that causes the fibroblasts in the tissue to produce new collagen, the addition of the nanostructures could immediately improve strength; preventing further injury and further strengthening the tissue once it has been healed by serving as a constant irritant to fibroblasts to produce structural ECM. In several in vitro studies, the cellular viability of rat patellar tendon fibroblasts in response to the different treatments was measured. After treatment, cell populations experienced increases in cellular viability. The results of these basic studies show the potential of prolotherapy and carbon nanostructures in ligament and tendon healing at a fundamental, cellular level. They also point to another mechanism for tissue healing after prolotherapy that is separate from the bodys wound healing cascade.