Benedikt L. Proffen

A comparative anatomical study of the human knee and six animal species

PURPOSE Animal models are an indispensable tool for developing and testing new clinical applications regarding the treatment of acute injuries and chronic diseases of the knee joint. Therefore, the purpose of this study was to compare the anatomy of the intra-articular structures of the human knee to species commonly used in large animal research studies. METHODS Fresh frozen cow (n=4), sheep (n=3), goat (n=4), dog (n=4), pig (n=5), rabbit (n=5), and human (n=4) cadaveric knees were used. Passive range of motion and intra-articular structure sizes of the knees were measured, the structure sizes normalized to the tibial plateau, and compared among the species. RESULTS Statistically significant differences in the range of motion and intra-articular structure sizes were found among all the species. Only the human knee was able to attain full extension. After normalization, only the pig ACL was significantly longer than the human counterpart. The tibial insertion site of the ACL was split by the anterior lateral meniscus attachment in the cow, sheep, and pig knees. The sheep PCL had two distinct tibial insertion sites, while all the other knees had only one. Furthermore, only in human knees, both lateral meniscal attachments were located more centrally than the medial meniscal attachments. CONCLUSIONS/CLINICAL RELEVANCE Despite the relatively preserved dimensions of the cruciate ligaments, menisci, and intercondylar notch amongst human and animals, structural differences in the cruciate ligament attachment sites and morphology of the menisci between humans and animals are important to consider when selecting an animal model.

Hypertrophy is induced during the in vitro chondrogenic differentiation of human mesenchymal stem cells by bone morphogenetic protein-2 and bone morphogenetic protein-4 gene transfer

IntroductionThe present study compares bone morphogenetic protein (BMP)-4 and BMP-2 gene transfer as agents of chondrogenesis and hypertrophy in human primary mesenchymal stem cells (MSCs) maintained as pellet cultures.MethodsAdenoviral vectors carrying cDNA encoding human BMP-4 (Ad.BMP-4) were constructed by cre-lox combination and compared to previously generated adenoviral vectors for BMP-2 (Ad.BMP-2), green fluorescent protein (Ad.GFP), or firefly luciferase (Ad.Luc). Cultures of human bone-marrow derived MSCs were infected with 5 × 102 viral particles/cell of Ad.BMP-2, or Ad.BMP-4, seeded into aggregates and cultured for three weeks in a defined, serum-free medium. Untransduced cells or cultures transduced with marker genes served as controls. Expression of BMP-2 and BMP-4 was determined by ELISA, and aggregates were analyzed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy.ResultsLevels of BMP-2 and BMP-4 in the media were initially 30 to 60 ng/mL and declined thereafter. BMP-4 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs as judged by lacuna formation, strong staining for proteoglycans and collagen type II, increased levels of GAG synthesis, and expression of mRNAs associated with the chondrocyte phenotype. However, BMP-4 modified aggregates showed a lower tendency to progress towards hypertrophy, as judged by expression of alkaline phosphatase, annexin 5, immunohistochemical staining for type X collagen protein, and lacunar size.ConclusionsBMP-2 and BMP-4 were equally effective in provoking chondrogenesis by primary human MSCs in pellet culture. However, chondrogenesis triggered by BMP-2 and BMP-4 gene transfer showed considerable evidence of hypertrophic differentiation, with, the cells resembling growth plate chondrocytes both morphologically and functionally. This suggests caution when using these candidate genes in cartilage repair.

Loss of extracellular matrix from articular cartilage is mediated by the synovium and ligament after anterior cruciate ligament injury

OBJECTIVE Post-traumatic osteoarthritis (PTOA) occurs after anterior cruciate ligament (ACL) injury. PTOA may be initiated by early expression of proteolytic enzymes capable of causing degradation of the articular cartilage at time of injury. This study investigated the production of three of these key proteases in multiple joint tissues after ACL injury and subsequent markers of cartilage turnover. METHODS ACL transection was performed in adolescent minipigs. Collagenase (MMP-1 and MMP-13) and aggrecanase (ADAMTS-4) gene expression changes were quantified in the articular cartilage, synovium, injured ligament, and the provisional scaffold at days 1, 5, 9, and 14 post-injury. Markers of collagen degradation (C2C), synthesis (CPII) and aggrecan synthesis (CS 846) were quantified in the serum and synovial fluid. Histologic assessment of the cartilage integrity (OARSI scoring) was also performed. RESULTS MMP-1 gene expression was upregulated in the articular cartilage, synovium and ligament after ACL injury. MMP-13 expression was suppressed in the articular cartilage, but upregulated 100-fold in the synovium and ligament. ADAMTS-4 was upregulated in the synovium and ligament but not in the articular cartilage. The concentration of collagen degradation fragments (C2C) in the synovial joint fluid nearly doubled in the first five days after injury. CONCLUSION We conclude that upregulation of genes coding for proteins capable of degrading cartilage ECM is seen within the first few days after ACL injury, and this response is seen not only in chondrocytes, but also in cells in the synovium, ligament and provisional scaffold.

Bio-Enhanced Repair of the Anterior Cruciate Ligament

Suture repair of the anterior cruciate ligament (ACL) has been widely abandoned in favor of ACL reconstruction, largely because of the high rates of failure and unreliability of the outcomes after suture repair. However, there have been recent basic science studies that suggest that combining a suture repair with a biological adjunct may improve the results of suture repair of the ACL, with several studies in large animal models showing equivalent strength of an ACL treated with bio-enhanced repaired to that of an ACL graft at 3, 6, and 12 months after surgery. In addition, the groups treated with bio-enhanced repair had significantly less osteoarthritis when compared with the animals undergoing ACL reconstruction. These findings have led to a renewed interest in bio-enhanced primary repair as a way to make repair of the ACL a viable option for a select group of patients in the future.

The Bridge-Enhanced Anterior Cruciate Ligament Repair (BEAR) Procedure An Early Feasibility Cohort Study

Background: This study assessed the safety of the newly developed bridge-enhanced anterior cruciate ligament (ACL) repair (BEAR), which involves suture repair of the ligament combined with a bioactive scaffold to bridge the gap between the torn ligament ends. As the intra-articular environment is complex in its response to implanted materials, this study was designed to determine whether there would be a significant rate of adverse reaction to the implanted scaffold. Hypothesis: The primary hypothesis was that the implanted scaffold would not result in a deep joint infection (arthrocentesis with positive culture) or significant inflammation (clinical symptoms justifying arthrocentesis but negative culture). The secondary hypotheses were that patients treated with BEAR would have early postoperative outcomes that were similar to patients treated with ACL reconstruction with an autologous hamstring graft. Study Design: Cohort study; Level of evidence, 2. Methods: A total of 20 patients were enrolled in this nonrandomized, first-in-human study. Ten patients received BEAR treatment and 10 received a hamstring autograft ACL reconstruction. The BEAR procedure was performed by augmenting a suture repair with a proprietary scaffold, the BEAR scaffold, placed in between the torn ends of the ACL at the time of suture repair. The BEAR scaffold is to our knowledge the only device that fills the gap between the torn ligament ends to have current Investigational Device Exemption approval from the Food and Drug Administration. Ten milliliters of autologous whole blood were added to the scaffold prior to wound closure. Outcomes were assessed at 3 months postoperatively. The outcomes measures included postoperative pain, muscle atrophy, loss of joint range of motion, and implant failure (designated by an International Knee Documentation Committee grade C or D Lachman test and/or an absence of continuous ACL tissue on magnetic resonance images). Results: There were no joint infections or signs of significant inflammation in either group. There were no differences between groups in effusion or pain, and no failures by Lachman examination criteria (BEAR, 8 grade A and 2 grade B; ACL reconstruction, 10 grade A). Magnetic resonance images from all of the BEAR and ACL-reconstructed patients demonstrated a continuous ACL or intact graft. In addition, hamstring strength at 3 months was significantly better in the BEAR group than in the hamstring autograft group (mean ± SD: 77.9% ± 14.6% vs 55.9% ± 7.8% of the contralateral side; P < .001). Conclusion: The results of this study suggest that the BEAR procedure may have a rate of adverse reactions low enough to warrant a study of efficacy in a larger group of patients.

A Normative Study of the Synovial Fluid Proteome from Healthy Porcine Knee Joints

Synovial fluid in an articulating joint contains proteins derived from the blood plasma and proteins that are produced by cells within the joint tissues, such as synovium, cartilage, ligament, and meniscus. The proteome composition of healthy synovial fluid and the cellular origins of many synovial fluid components are not fully understood. Here, we present a normative proteomics study using porcine synovial fluid. Using our optimized method, we identified 267 proteins with high confidence in healthy synovial fluid. We also evaluated mRNA expression data from tissues that can contribute to the synovial fluid proteome, including synovium, cartilage, blood, and liver, to better estimate the relative contributions from these sources to specific synovial fluid components. We identified 113 proteins in healthy synovial fluid that appear to be primarily derived from plasma transudates, 37 proteins primarily derived from synovium, and 11 proteins primarily derived from cartilage. Finally, we compared the identified synovial fluid proteome to the proteome of human plasma, and we found that the two body fluids share many similarities, underlining the detected plasma derived nature of many synovial fluid components. Knowing the synovial fluid proteome of a healthy joint will help to identify mechanisms that cause joint disease and pathways involved in disease progression.

Mesenchymal Stem Cells from the Retropatellar Fat Pad and Peripheral Blood Stimulate ACL Fibroblast Migration, Proliferation, and Collagen Gene Expression

Mesenchymal stem cells (MSCs) have been of recent interest as adjuncts for ligament repair. However, the effect of these cells on the resident ligament fibroblasts has not yet been defined. In this study, we hypothesized that co-culture of MSCs and ligament fibroblasts would result in increases in the proliferative rate of the ligament fibroblasts and their expression of collagen-related genes, as well as differentiation of the MSCs down a fibroblastic pathway. In addition, we hypothesized that these effects would be dependent on the source of the MSCs. Porcine MSCs were isolated from both the retro-patellar fat pad (ADSCs) and the peripheral blood (PBMCs) and co-cultured with porcine anterior cruciate ligament (ACL) fibroblasts. Fibroblast migration, proliferation, and collagen gene expression were evaluated at time points up to 14 days. ADSCs had a greater effect on stimulating ACL-fibroblast proliferation and procollagen production, while PBMCs were more effective in stimulating ligament fibroblast migration. In addition, co-culture with the ACL fibroblasts led to significant increases in collagen gene expression for ADSCs, suggesting a differentiation of these cells down a fibroblastic pathway during the co-culture period. This was not seen for the PBMCs. Thus, the effects of MSCs on in situ ACL fibroblasts were found to be source dependent, and the choice of MSC source should take into account the different performance characteristic of each type of MSC.

Addition of Autologous Mesenchymal Stem Cells to Whole Blood for Bioenhanced ACL Repair Has No Benefit in the Porcine Model

Background: Coculture of mesenchymal stem cells (MSCs) from the retropatellar fat pad and peripheral blood has been shown to stimulate anterior cruciate ligament (ACL) fibroblast proliferation and collagen production in vitro. Current techniques of bioenhanced ACL repair in animal studies involve adding a biologic scaffold, in this case an extracellular matrix–based scaffold saturated with autologous whole blood, to a simple suture repair of the ligament. Whether the enrichment of whole blood with MSCs would further improve the in vivo results of bioenhanced ACL repair was investigated. Hypothesis: The addition of MSCs derived from adipose tissue or peripheral blood to the blood–extracellular matrix composite, which is used in bioenhanced ACL repair to stimulate healing, would improve the biomechanical properties of a bioenhanced ACL repair after 15 weeks of healing. Study Design: Controlled laboratory study. Methods: Twenty-four adolescent Yucatan mini-pigs underwent ACL transection followed by (1) bioenhanced ACL repair, (2) bioenhanced ACL repair with the addition of autologous adipose-derived MSCs, and (3) bioenhanced ACL repair with the addition of autologous peripheral blood derived MSCs. After 15 weeks of healing, the structural properties of the ACL (yield load, failure load, and linear stiffness) were measured. Cell and vascular density were measured in the repaired ACL via histology, and its tissue structure was qualitatively evaluated using the advanced Ligament Maturity Index. Results: After 15 weeks of healing, there were no significant improvements in the biomechanical or histological properties with the addition of adipose-derived MSCs. The only significant change with the addition of peripheral blood MSCs was an increase in knee anteroposterior laxity when measured at 30° of flexion. Conclusion: These findings suggest that the addition of adipose or peripheral blood MSCs to whole blood before saturation of an extracellular matrix carrier with the blood did not improve the functional results of bioenhanced ACL repair after 15 weeks of healing in the pig model. Clinical Relevance: Whole blood represents a practical biologic additive to ligament repair, and any other additive (including stem cells) should be demonstrated to be superior to this baseline before clinical use is considered.

Immediate Administration of Intraarticular Triamcinolone Acetonide After Joint Injury Modulates Molecular Outcomes Associated With Early Synovitis

To test whether intraarticular corticosteroid injection mitigates injury‐induced synovitis and collagen degradation after anterior cruciate ligament transection (ACLT) and to characterize the synovial response using a functional genomics approach in a preclinical model of posttraumatic osteoarthritis.

Histological Predictors of Maximum Failure Loads Differ Between the Healing ACL and ACL Grafts After 6 and 12 Months In Vivo

Background: Bioenhanced anterior cruciate ligament (ACL) repair, where the suture repair is supplemented with a biological scaffold, is a promising novel technique to stimulate healing after ACL rupture. However, the histological properties of a successfully healing ACL and how they relate to the mechanical properties have not been fully described. Purpose: To determine which histological features best correlate with the mechanical properties of the healing ACL repairs and ACL grafts in a porcine model at 6 and 12 months after injury. Study Design: Controlled laboratory study. Methods: A total of 48 Yucatan mini-pigs underwent ACL transection followed by: (1) conventional ACL reconstruction with bone–patellar tendon–bone (BPTB) allograft, (2) bioenhanced ACL reconstruction with BPTB allograft using a bioactive scaffold, or (3) bioenhanced ACL repair using the same bioactive scaffold. After 6 and 12 months of healing, structural properties of the ACL or graft (yield and failure load, linear stiffness) were measured. Following mechanical testing, ACL specimens were histologically analyzed for cell and vascular density and qualitatively assessed using the advanced Ligament Maturity Index. Results: After 6 months of healing, the cellular organization subscore was most predictive of yield load (r 2 = 0.98), maximum load (r 2 = 0.89), and linear stiffness (r 2 = 0.95) of the healing ACL, while at 12 months, the collagen subscore (r 2 = 0.68) became the best predictor of maximum load. For ACL grafts, the reverse was true, with the collagen subscore predictive of yield and maximum loads at 6 months (r 2 = 0.55) and graft cellularity predictive of maximum load of the graft at 12 months (r 2 = 0.50). Conclusion: These findings suggest there may be key biological differences in development and maintenance of ACL tissue after repair or reconstruction, with early ligament function dependent on cellular population of the repair but early graft function dependent on the maintenance of organized collagen. Clinical Relevance: Bioenhanced ACL repair shows promising potential as an alternative clinical treatment for ACL injury. This study contributes to the understanding of the cellular contribution to mechanical characteristics of the healing ACL in both repaired and reconstructed ACLs.