Human fascia lata ECM scaffold augmented with immobilized hyaluronan: inflammatory response and remodeling in the canine body wall and shoulder implantation sites.

We postulate that immobilization of tyramine-substituted hyaluronan (THA) into an extracellular matrix (ECM) scaffold may be a strategy to promote an anti-inflammatory response to the ECM. Further, we posit that the implantation site could influence the inflammatory response and remodeling of an ECM scaffold. Eight beagles underwent implantation of fascia ECM grafts, treated with either immobilized low molecular weight (57 kDa) THA or water only, in both the shoulder injury and body wall sites. Dogs were euthanized at 12 weeks and fascia grafts harvested en bloc for histology. Grafts implanted at the body wall had significantly higher inflammatory cell infiltrate and vascularity, and significantly lower retardance (collagen density), than grafts at the shoulder, suggestive of a more intense, persistent, and perhaps degradative inflammatory and remodeling response at the body wall than shoulder injury site in the canine model. However, the presence of immobilized low MW THA had no effect on the inflammation response or remodeling of fascia ECM compared to water-treated controls. Importantly, these results suggest that the inflammatory response and remodeling of biomaterial implants depends on the location of implantation and therefore our animal models need to be carefully chosen. Further, the potential anti-inflammatory advantages of hyaluronan (HA) in wound healing do not appear to be realized when presenting it to the host as non-degradable hydrogel even if its capacity for binding HA binding protein is maintained. Further study treating ECM with uncross-linked (free) HA or immobilized low MW THA as a means to deliver free HA or other biomolecules to a surgical repair site is warranted.

Host response to xenograft ECM implantation is not different between the shoulder and body wall sites in the rat model.

In the context of tendon and ligament repair, mechanical loading and the presence of joint synovial fluid are known to profoundly influence the form and function of the repair tissue and potentially the host response to biomaterials. Previously, we demonstrated that a xenograft extra cellular matrix (ECM) scaffold implanted in the rat shoulder elicited a unique host response from that seen in the body wall. However, the host response to xenografts implanted in shoulders with a tendon/capsule injury was not different from xenografts implanted in shoulders with no injury. In the current study, we hypothesized that varying clinically relevant surgical and environmental factors would introduce significant differences in host response to xenograft implantation at the shoulder. Contrary to our hypothesis, we found no significant differences in host response between any shoulder implantation conditions or between shoulder and body wall implantation in the rat model. These findings suggest that there is no advantage to using an orthotopic shoulder model to investigate the host response to rotator cuff scaffold materials in the rat model, and due to the insensitivity of its host response to various clinically relevant surgical conditions, may suggest that the rat does not provide a surrogate for directly translating the host response to biomaterials to the human application.

Effect of implantation site and injury condition on host response to human-derived fascia lata ECM in a rat model.

The host response and remodeling of ECM scaffolds are believed to be critical determinants of success or failure in repair or reconstructive procedures. Host response has been investigated in subcutaneous or abdominal wall implantation models. The extent to which evaluation of the host response to ECM intended for tendon or ligament repair should be performed in an orthotopic site is not known. This study compared the host response to human-derived fascia lata ECM among various implantation sites in the rat model. Results showed that a xenograft in the rat shoulder does not exhibit a different host response at 7 days from xenograft in the body wall, suggesting that either site may be appropriate to study the early host response to biologic grafts as well as the effect of various treatments aimed to modify the early host response. By 28 days, a xenograft in the rat shoulder does elicit a unique host response from that seen in the body wall. Therefore, it may be more appropriate to use an orthotopic shoulder model for investigating the long-term host response and remodeling of biologic grafts to be used for rotator cuff repair.

Changes in gene expression of individual matrix metalloproteinases differ in response to mechanical unloading of tendon fascicles in explant culture.

Immobilization of the tendon and ligament has been shown to result in a rapid and significant decrease in material properties. It has been proposed that tissue degradation leading to tendon rupture or pain in humans may also be linked to mechanical unloading following focal tendon injury. Hence, understanding the remodeling mechanism associated with mechanical unloading has relevance for the human conditions of immobilization (e.g., casting), delayed repair of tendon ruptures, and potentially overuse injuries as well. This is the first study to investigate the time course of gene expression changes associated with tissue harvest and mechanical unloading culture in an explant model. Rat tail tendon fascicles were harvested and placed in culture unloaded for up to 48 h and then evaluated using qRT-PCR for changes in two anabolic and four catabolic genes at 12 time points. Our data demonstrates that Type I Collagen, Decorin, Cathepsin K, and MMP2 gene expression are relatively insensitive to unloaded culture conditions. However, changes in both MMP3 and MMP13 gene expression are rapid, dramatic, sustained, and changing during at least the first 48 h of unloaded culture. This data will help to further elucidate the mechanism for the loss of mechanical properties associated with mechanical unloading in tendon.

Regional variability, processing methods, and biophysical properties of human fascia lata extracellular matrix.

This study aims to assess the regional variability, processing methods, mechanical, biochemical, and cellular properties of human fascia lata as a scaffold for soft tissue repair and tissue engineering applications. Ten pairs of fascia lata (donor age 18-55) were used. One fascia patch from each pair was used to assess the geometric and biomechanical variability of fresh fascia. The other from each pair was subjected to 1 of 2 allograft processing methods: antibiotic soak alone or acellularization plus antibiotic soak. Stiffness, modulus, hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan (CSDS GAG), and DNA content were quantified in fascia from fresh and treated groups. The effect of location was not significant for thickness or stiffness within a 6 x 12 cm2 region of the iliotibial tract of fresh human fascia lata. Processing did not significantly change the stiffness, modulus, or CSDS GAG content of fascia ECM. However, hydroxyproline (collagen) content is significantly reduced in acellularized fascia, probably reflecting a removal of soluble collagen during the treatment (p < 0.02). Processing reduced the DNA content of fresh fascia approximately 10-fold (p < 0.001). The mechanical, chemical and ultrastructural similarities between fascia lata and tendon may make fresh or processed fascia an attractive ECM scaffold for soft tissue, particularly tendon, repair.

Commercial extracellular matrix scaffolds for rotator cuff tendon repair. Biomechanical, biochemical, and cellular properties.

BACKGROUND: We are not aware of any in vitro study comparing the biomechanical, biochemical, and cellular properties of commercial extracellular matrix materials marketed for rotator cuff tendon repair. In this study, the properties of GraftJacket, TissueMend, Restore, and CuffPatch were quantified and compared with each other. The elastic moduli were also compared with that of normal canine infraspinatus tendon. METHODS: Samples were tested from different manufacturing lots of four materials: GraftJacket (ten lots), TissueMend (six), Restore (ten), and CuffPatch (six). The Kruskal-Wallis test was used to compare thickness, stiffness, and modulus as well as hydroxyproline, chondroitin/dermatan sulfate glycosaminoglycan, hyaluronan, and DNA contents among these matrices. The moduli of the extracellular matrices were also compared with those of normal canine infraspinatus tendon. RESULTS: All four extracellular matrices required 10% to 30% stretch before they began to carry substantial load. Their maximum moduli were realized in their linear region at 30% to 80% strain. The elastic moduli of all four commercial matrices were an order of magnitude lower than that of canine infraspinatus tendon. TissueMend had significantly higher DNA content than the other three matrices (p<0.0001), although both Restore and GraftJacket also had measurable amounts of DNA. CONCLUSIONS: Our data demonstrate chemical and mechanical differences among the four commercial extracellular matrices that we evaluated. Probably, the source (dermis or small intestine submucosa), species (human, porcine, or bovine), age of the donor (fetal or adult), and processing of these matrices all contribute to the unique biophysical properties of the delivered product. The biochemical composition of commercial extracellular matrices is similar to that of tendon. However, the elastic moduli of these materials are an order of magnitude lower than that of tendon, suggesting a limited mechanical role in augmentation of tendon repair.

Cell tracking velocimetry as a tool for defining saturation binding of magnetically conjugated antibodies.

BACKGROUND: Continuous flow immunomagnetic separation is an attractive alternative to current batch mode immunomagnetic separation methods because it is capable of high sorting speeds at mild cell conditions, and grants the operator better control of separation process. The control of the separation is dependent on knowledge of the amount of magnetic label attached to the cell (magnetic labeling intensity), however. Determination of the magnetic labeling is accomplished by measuring cell magnetophoretic mobility using a newly developed technique of Cell Tracking Velocimetry (CTV). METHODS: Flow cytometry was used to define the antibody binding characteristics of a fluorescently tagged primary antibody. Subsequently, CTV was used to measure antibody-binding characteristics of a magnetically tagged secondary antibody. RESULTS: The results of this study show that CTV is capable of providing valuable information concerning the cell labeling by magnetically tagged antibodies. It was demonstrated that the magnetically conjugated antibody binding curve exhibits the same exponential increase to saturation characteristics as that seen with the fluorescently tagged antibody. Further, it was shown that the intensity of the secondary magnetic labeling is directly proportional to the intensity of the primary fluorescent label. CONCLUSIONS: CTV is an accurate tool for evaluation of magnetically conjugated antibodies. The ability to determine the intensity of magnetic labeling is necessary for the development of continuous flow immunomagnetic separations based on cell magnetophoresis.