Osteoarthritic meniscal entheses exhibit altered collagen fiber orientation.

PURPOSE/AIM: The knee menisci are vital for maintaining the stability of the joint, allowing for force distribution, and protecting the underlying articular cartilage during loading. Each meniscus is attached to the underlying bone via two ligamentous entheses composed of collagen fibers that are continuous throughout all four zones of the attachment: ligament, uncalcified fibrocartilage, calcified fibrocartilage, and subchondral bone. The collagen fibers of the meniscal entheses are important for proper functionality of the entheses, particularly in preventing meniscal extrusion which is a common hallmark of osteoarthritis. The goal of this work was to assess changes in collagen fiber orientation present in osteoarthritic knee joints. MATERIALS AND METHODS: Entheses were harvested from patients undergoing total knee arthroplasties and prepared histological sections were stained with picrosirius red to identify collagen fiber angle and fiber deviation. RESULTS: In the calcified fibrocartilage the collagen fibers of the lateral anterior enthesis inserted at significantly (p < 0.1) shallower angles, and the fiber deviation was significantly (p < 0.1) less compared to the lateral posterior enthesis. These differences in the calcified fibrocartilage may occur as an adaptation to loading regimes of the osteoarthritic joint. When compared to the collagen fiber orientation of healthy entheses, collagen fibers in osteoarthritic tissue inserted at shallower insertion angles and demonstrated higher levels of deviation. CONCLUSIONS: Changes to meniscal enthesis collagen fiber orientation with end stage osteoarthritis could offer an explanation for the change in functionality of diseased tissue and may contribute to meniscal extrusion and ultimately the degeneration of articular cartilage.

Improving the Intercellular Uptake and Osteogenic Potency of Calcium Phosphate via Nanocomplexation with the RALA Peptide.

Calcium phosphate-base materials (e.g., alpha tri-calcium phosphate (α-TCP)) have been shown to promote osteogenic differentiation of stem/progenitor cells, enhance osteoblast osteogenic activity and mediate in vivo bone tissue formation. However, variable particle size and hydrophilicity of the calcium phosphate result in an extremely low bioavailability. Therefore, an effective delivery system is required that can encapsulate the calcium phosphate, improve cellular entry and, consequently, elicit a potent osteogenic response in osteoblasts. In this study, collagenous matrix deposition and extracellular matrix mineralization of osteoblast lineage cells were assessed to investigate osteogenesis following intracellular delivery of α-TCP nanoparticles. The nanoparticles were formed via condensation with a novel, cationic 30 mer amphipathic peptide (RALA). Nanoparticles prepared at a mass ratio of 5:1 demonstrated an average particle size of 43 nm with a zeta potential of +26 mV. The average particle size and zeta potential remained stable for up to 28 days at room temperature and across a range of temperatures (4-37 °C). Cell viability decreased 24 h post-transfection following RALA/α-TCP nanoparticle treatment; however, recovery ensued by Day 7. Immunocytochemistry staining for Type I collagen up to Day 21 post-transfection with RALA/α-TCP nanoparticles (NPs) in MG-63 cells exhibited a significant enhancement in collagen expression and deposition compared to an untreated control. Furthermore, in porcine mesenchymal stem cells (pMSCs), there was enhanced mineralization compared to α-TCP alone. Taken together these data demonstrate that internalization of RALA/α-TCP NPs elicits a potent osteogenic response in both MG-63 and pMSCs.

A Hydrogel Meniscal Replacement: Knee Joint Pressure and Distribution in an Ovine Model Compared to Native Tissue.

Pressure distribution of the native ovine knee meniscus was compared to a medial meniscectomy and three treatment conditions including a suture reattachment of the native tissue, an allograft, and a novel thermoplastic elastomer hydrogel (TPE) construct. The objective of this study was to assess the efficacy of a novel TPE hydrogel construct at restoring joint pressure and distribution. Limbs were loaded in uniaxial compression at 45°, 60°, and 75° flexion and from 0 to 181 kg. The medial meniscectomy decreased contact area by approximately 50% and doubled the mean and maximum pressure reading for the medial hemijoint. No treatment condition tested within this study was able to fully restore medial joint contact area and pressures to the native condition. A decrease in lateral contact area and increase in pressures with the meniscectomy was also seen; and to some degree, all reattachment and replacement conditions including the novel TPE hydrogel replacement helped to restore lateral pressures. Although the TPE construct did not perform as well as hoped in the medial compartment, it performed as well as, if not better, than the other reattachment and replacement options in the lateral. Further work is necessary to determine the best anchoring and attachment methods.

Mechanical Properties and Cell Compatibility of Agarose Hydrogels Containing Proteoglycan Mimetic Graft Copolymers.

Proteoglycans have vital biochemical and biomechanical functions. Their proteolytic degradation results in loss of these functions. We have previously reported nonprotein proteoglycan-mimetic graft copolymers that stabilize and deliver growth factors and are not subject to proteases. Here we expand our investigation of these proteoglycan mimics by also investigating their effects on hydrogel mechanical properties. Four polysaccharide side chains, chondroitin sulfate, heparin, dextran, and dextran sulfate, are each grafted to a hyaluronan backbone. The polysaccharides and graft copolymers are added to agarose hydrogels. Cyclic compression and stress relaxation tests reveal how the addition of the polysaccharides and graft copolymers influence hydrogel modulus. Cells encapsulated in agarose hydrogels containing chondroitin sulfate and the chondroitin sulfate graft copolymer have decreased cell viability and metabolic activity compared to cells in unmodified agarose hydrogels. These multifunctional additives can be used to improve both the biochemistry and biomechanics of materials, warranting further optimization to overcome the potentially negative effects these may have on cell viability and activity.

* Hierarchically Structured Electrospun Scaffolds with Chemically Conjugated Growth Factor for Ligament Tissue Engineering.

The anterior cruciate ligament (ACL) of the knee is vital for proper joint function and is commonly ruptured during sports injuries or car accidents. Due to a lack of intrinsic healing capacity and drawbacks with allografts and autografts, there is a need for a tissue-engineered ACL replacement. Our group has previously used aligned sheets of electrospun polycaprolactone nanofibers to develop solid cylindrical bundles of longitudinally aligned nanofibers. We have shown that these nanofiber bundles support cell proliferation and elongation and the hierarchical structure and material properties are similar to the native human ACL. It is possible to combine multiple nanofiber bundles to create a scaffold that attempts to mimic the macroscale structure of the ACL. The goal of this work was to develop a hierarchical bioactive scaffold for ligament tissue engineering using connective tissue growth factor (CTGF)-conjugated nanofiber bundles and evaluate the behavior of mesenchymal stem cells (MSCs) on these scaffolds in vitro and in vivo. CTGF was immobilized onto the surface of individual nanofiber bundles or scaffolds consisting of multiple nanofiber bundles. The conjugation efficiency and the release of conjugated CTGF were assessed using X-ray photoelectron spectroscopy, assays, and immunofluorescence staining. Scaffolds were seeded with MSCs and maintained in vitro for 7 days (individual nanofiber bundles), in vitro for 21 days (scaled-up scaffolds of 20 nanofiber bundles), or in vivo for 6 weeks (small scaffolds of 4 nanofiber bundles), and ligament-specific tissue formation was assessed in comparison to non-CTGF-conjugated control scaffolds. Results showed that CTGF conjugation encouraged cell proliferation and ligament-specific tissue formation in vitro and in vivo. The results suggest that hierarchical electrospun nanofiber bundles conjugated with CTGF are a scalable and bioactive scaffold for ACL tissue engineering.

Mechanical properties and cellular response of novel electrospun nanofibers for ligament tissue engineering: Effects of orientation and geometry.

Electrospun nanofibers are a promising material for ligamentous tissue engineering, however weak mechanical properties of fibers to date have limited their clinical usage. The goal of this work was to modify electrospun nanofibers to create a robust structure that mimics the complex hierarchy of native tendons and ligaments. The scaffolds that were fabricated in this study consisted of either random or aligned nanofibers in flat sheets or rolled nanofiber bundles that mimic the size scale of fascicle units in primarily tensile load bearing soft musculoskeletal tissues. Altering nanofiber orientation and geometry significantly affected mechanical properties; most notably aligned nanofiber sheets had the greatest modulus; 125% higher than that of random nanofiber sheets; and 45% higher than aligned nanofiber bundles. Modifying aligned nanofiber sheets to form aligned nanofiber bundles also resulted in approximately 107% higher yield stresses and 140% higher yield strains. The mechanical properties of aligned nanofiber bundles were in the range of the mechanical properties of the native ACL: modulus=158±32MPa, yield stress=57±23MPa and yield strain=0.38±0.08. Adipose derived stem cells cultured on all surfaces remained viable and proliferated extensively over a 7 day culture period and cells elongated on nanofiber bundles. The results of the study suggest that aligned nanofiber bundles may be useful for ligament and tendon tissue engineering based on their mechanical properties and ability to support cell adhesion, proliferation, and elongation.

Assessment of cortical and trabecular bone changes in two models of post-traumatic osteoarthritis.

Subchondral bone is thought to play a significant role in the initiation and progression of the post-traumatic osteoarthritis. The goal of this study was to document changes in tibial and femoral subchondral bone that occur as a result of two lapine models of anterior cruciate ligament injury, a modified ACL transection model and a closed-joint traumatic compressive impact model. Twelve weeks post-injury bones were scanned via micro-computed tomography. The subchondral bone of injured limbs from both models showed decreases in bone volume and bone mineral density. Surgical transection animals showed significant bone changes primarily in the medial hemijoint of femurs and tibias, while significant changes were noted in both the medial and lateral hemijoints of both bones for traumatic impact animals. It is believed that subchondral bone changes in the medial hemijoint were likely caused by compromised soft tissue structures seen in both models. Subchondral bone changes in the lateral hemijoint of traumatic impact animals are thought to be due to transmission of the compressive impact force through the joint. The joint-wide bone changes shown in the traumatic impact model were similar to clinical findings from studies investigating the progression of osteoarthritis in humans.