Biology of anterior cruciate ligament injury and repair: Kappa delta ann doner vaughn award paper 2013.

Anterior cruciate ligament (ACL) injuries are currently treated by removing the injured ligament and replacing it with a tendon graft. Recent studies have examined alternative treatment methods, including repair and regeneration of the injured ligament. In order to make such an approach feasible, a basic understanding of ACL biology and its response to injury is needed. Identification of obstacles to native ACL healing can then be identified and potentially resolved using tissue engineering strategies-first, with in vitro screening assays, and then with in vivo models of efficacy and safety. This Perspectives paper outlines this path of discovery for optimizing ACL healing using a bio-enhanced repair technique. This journey required constructing indices of the functional tissue response, pioneering physiologically based methods of biomechanical testing, developing, and validating clinically relevant animal models, and creating and optimizing translationally feasible scaffolds, surgical techniques, and biologic additives. Using this systematic translational approach, "bio-enhanced" ACL repair has been advanced to the point where it may become an option for future treatment of acute ACL injuries and the prevention of subsequent post-traumatic osteoarthritis associated with this injury.

Platelets and plasma proteins are both required to stimulate collagen gene expression by anterior cruciate ligament cells in three-dimensional culture.

Collagen-platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis.

Interspecies variation in the fibroblast distribution of the anterior cruciate ligament.

BACKGROUND: The future of the treatment of a ruptured anterior cruciate ligament is likely to involve cell-based therapies. These therapies are intrinsically dependent on the cellular distributions in the ligament. Thus, when selecting an animal model for testing of these new treatment methods, it is important to select a model that has similar cellular distributions to that of the normal human anterior cruciate ligament. HYPOTHESIS: There are interspecies differences in the histology of the anterior cruciate ligament. STUDY DESIGN: A descriptive histological study comparing the cell and vessel distribution of normal human anterior cruciate ligaments with that of 3 animal models and anterior cruciate ligaments from osteoarthritic human knees. METHODS: The histology of each of the anterior cruciate ligament sources was quantified in terms of cell number density, expression of alpha-smooth muscle actin, blood vessel density, and cell nuclear morphology using standardized histomorphometric techniques. RESULTS: The normal human anterior cruciate ligament was similar to the canine anterior cruciate ligament and the anterior cruciate ligament from patients with osteoarthritis with respect to cell density, blood vessel density, and cell nuclear shape. The normal anterior cruciate ligament had significantly fewer vessels than the bovine anterior cruciate ligament and rounder cells than the bovine and ovine anterior cruciate ligaments. CONCLUSIONS: There is significant interspecies variation in the histology of the anterior cruciate ligament, with the canine anterior cruciate ligament most similar to the human anterior cruciate ligament. CLINICAL RELEVANCE: This finding may have an effect on the accuracy of testing of new, cell-based treatments for the ruptured anterior cruciate ligament, such as guided tissue regeneration.

Enhanced repair of the anterior cruciate ligament by in situ gene transfer: evaluation in an in vitro model.

The inability of the ruptured anterior cruciate ligament (ACL) of the knee joint to heal spontaneously presents numerous clinical problems. Here we describe a novel, gene-based approach to augment ACL healing. It is based upon the migration of cells from the ruptured ends of the ligament into a collagen hydrogel laden with recombinant adenovirus. Cells entering the gel become transduced by the vector, which provides a basis for the local synthesis of gene products that aid repair. Monolayers of bovine ACL cells were readily transduced by first-generation, recombinant adenovirus, and transgene expression remained high after the cells were incorporated into collagen hydrogels. Using an in vitro model of ligament repair, cells migrated from the cut ends of the ACL into the hydrogel and were readily transduced by recombinant adenovirus contained within it. The results of experiments in which GFP was used as the transgene suggest highly efficient transduction of ACL cells in this manner. Moreover, during a 21-day period GFP+ cells were observed more than 6 mm from the severed ligament. This distance is ample for the projected clinical application of this technology. In response to TGF-beta1 as the transgene, greater numbers of ACL cells accumulated in the hydrogels, where they deposited larger amounts of type III collagen. These data confirm that it is possible to transduce ACL cells efficiently in situ as they migrate from the ruptured ACL, that transduction does not interfere with the cells' ability to migrate distances necessary for successful repair, and that ACL cells will respond in a suitable manner to the products of the transgenes they express. This permits optimism over a possible clinical use for this technology.

The death of articular chondrocytes after intra-articular fracture in humans.

BACKGROUND: In this study, we wished to determine whether chondrocyte death occurs after intra-articular fracture and might thus contribute to the development of posttraumatic arthritis. METHODS: Articular fracture fragments were obtained from 30 patients and the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay was used to identify dead or dying cells. RESULTS: Dead or dying cells were identified in all fracture fragments, with an average rate of cell death of 35%. Rates of cell death greater than 90% were seen in 4 of the 30 specimens studied. CONCLUSION: Although rates of apoptosis in osteoarthritis have been reported to average 15%, the rate in this fracture population was more than twice this value (35%). The high rate of cell death noted here may help explain the occurrence of posttraumatic arthritis even in anatomically fixed intra-articular fractures.

Cell outgrowth from the human ACL in vitro: regional variation and response to TGF-beta1.

One of the new methods being developed to stimulate healing of the human anterior cruciate ligament (ACL) after rupture is the implantation of a biodegradable scaffold which the host cells invade, populate and remodel. One of the cellular behaviors critical to the success of this method is cell outgrowth from the ligament remnants onto an adjacent scaffold. As morphological differences have been previously reported in the proximal and distal human ACL, the primary aim of this study was to determine if the cells from the proximal and distal ACL had different outgrowth behaviors as well. A second aim was to determine whether TGF-beta1, reported to be a mitogen for fibroblasts, would affect the outgrowth behaviors from the proximal and distal ACL. To achieve these aims, explants from both the proximal and distal human ACL were placed into culture under various conditions and outgrowth measured for 42 days. In explants cultured with 10% fetal bovine serum (high serum group) the explants from the proximal ACL had an earlier start of outgrowth than the distal explants (p < 0.015), and outgrowth rates were similar in the two groups. In explants cultured with 2% fetal bovine serum (low serum group), the explants from the proximal ACL had an earlier start to outgrowth (p < 0.003) as well as a faster rate of outgrowth (p < 0.004) than the distal explants. The addition of TGF-beta1 to the low serum cultures significantly slowed the rate of outgrowth from both groups of ACL explants (p < 0.025). These results suggest that outgrowth behaviors are different in the proximal and distal human ACL, and that TGB-beta1 has an inhibitory effect on cell outgrowth from ACL explants. However, this study is only a first step, and additional experiments are needed to further optimize tissue engineering parameters for enhancement of the repair of the ACL.