Removal of the infrapatellar fat pad and associated synovium benefits female guinea pigs in the Dunkin Hartley model of idiopathic osteoarthritis.

Background: Several tissues contribute to the onset and advancement of knee osteoarthritis (OA). One tissue type that is worthy of closer evaluation, particularly in the context of sex, is the infrapatellar fat pad (IFP). We previously demonstrated that removal of the IFP had short-term beneficial effects for a cohort of male Dunkin-Hartley guinea pigs. The present project was designed to elucidate the influence of IFP removal in females of this OA-prone strain. It was hypothesized that resection of the IFP would reduce the development of OA in knees of a rodent model predisposed to the disease. Methods: Female guinea pigs (n=16) were acquired at an age of 2.5 months. Surgical removal of the IFP and associated synovium complex (IFP/SC) was executed at 3 months of age. One knee had the IFP/SC resected; a comparable sham surgery was performed on the contralateral knee. All animals were subjected to voluntary enclosure monitoring and dynamic weight-bearing, as well as compulsory treadmill-based gait analysis monthly; baseline data was collected prior to surgery. Guinea pigs were euthanized at 7 months. Knees from eight animals were evaluated via histology, mRNA expression, and immunohistochemistry (IHC); knees from the remaining eight animals were allocated to microcomputed tomography (microCT), biomechanical analyses (whole joint testing and indentation relaxation testing), and atomic absorption spectroscopy (AAS). Results: Fibrous connective tissue (FCT) replaced the IFP/SC. Mobility/gait data indicated that unilateral IFP/SC removal did not affect bilateral hindlimb movement. MicroCT demonstrated that osteophytes were not a significant feature of OA in this sex; however, trabecular thickness (TbTh) in medial femorae decreased in knees containing the FCT. Histopathology scores were predominantly influenced by changes in the lateral tibia, which demonstrated that histologic signs of OA were increased in knees containing the native IFP/SC versus those with the FCT. Similarly, indentation testing demonstrated higher instantaneous and equilibrium moduli in the lateral tibial articular cartilage of control knees with native IFPs. AAS of multiple tissue types associated with the knee revealed that zinc was the major trace element influenced by removal of the IFP/SC. Conclusions: Our data suggest that the IFP/SC is a significant component driving knee OA in female guinea pigs and that resection of this tissue prior to disease has short-term benefits. Specifically, the formation of the FCT in place of the native tissue resulted in decreased cartilage-related OA changes, as demonstrated by reduced Osteoarthritis Research Society International (OARSI) histology scores, as well as changes in transcript, protein, and cartilage indentation analyses. Importantly, this model provides evidence that sex needs to be considered when investigating responses and associated mechanisms seen with this intervention.

Early removal of the infrapatellar fat pad/synovium complex beneficially alters the pathogenesis of moderate stage idiopathic knee osteoarthritis in male Dunkin Hartley guinea pigs.

BACKGROUND: The infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of the IFP and its associated synovium (IFP/synovium complex or IFP/SC) on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of this unit in a rodent model of naturally-occurring disease. METHODS: Male Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP/SC removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity). RESULTS: Fibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP/SC removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP/SC-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFP/SCs. This was verified via IHC for two key inflammatory agents. The medial articular cartilage in knees with native IFP/SCs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus. DISCUSSION/CONCLUSION: Formation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP/SC may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Loosening of Posteromedial Meniscal Root Repairs Affects Knee Mechanics: A Finite Element Study.

Meniscal root repairs are susceptible to unrecoverable loosening that may displace the meniscus from the initial position reduced during surgery. Despite this, the effects of a loosened meniscal root repair on knee mechanics are unknown. We hypothesized that anatomic root repairs without loosening would restore knee mechanics to the intact condition better than loosened anatomic root repairs, but that loosened repairs would restore mechanics better than untreated meniscal root tears. Finite element knee models were used to evaluate changes in cartilage and meniscus mechanics due to repair loosening. The mechanical response from loosened anatomic root repairs was compared to anatomic repairs without loosening and untreated root tears. All conditions were evaluated at three flexion angles, 0 deg, 30 deg, and 60 deg, and a compressive force of 1000 N to simulate return-to-activity loading. The two-simple suture method was represented within the models to simulate posteromedial meniscal root repairs and the loosening of repairs was derived from previous biomechanical experimental data. Loosening decreased hoop stresses throughout the meniscus, increased posterior extrusion, and shifted loading through the meniscus-cartilage region to the cartilage-cartilage region compared to the anatomic root repair without loosening. Despite differences between repairs and loosened repairs, the changes from loosened repairs more closely resembled the anatomic repair without loosening than the untreated root repair condition. Therefore, meniscal root repairs are susceptible to loosening that will prevent a successful initial repair from remaining in the intended position and will alter cartilage and meniscus mechanics, although repairs that loosen appear better than leaving tears untreated.

Evaluating the Efficacy of Combined P188 Treatment and Surgical Intervention in Preventing Post-Traumatic Osteoarthritis Following a Traumatic Knee Injury.

Previous studies have shown that reconstructive surgery alone following injury to the anterior cruciate ligament (ACL) does not prevent the development of post-traumatic osteoarthritis (PTOA). Poloxamer 188 (P188) has been shown to prevent cell death following trauma in both articular cartilage and meniscal tissue. This study aims to test the efficacy of single or multiple administrations of P188 in conjunction with reconstructive surgery to help prevent or delay the onset of the disease. Thirty skeletally mature rabbits underwent closed-joint trauma that resulted in ACL rupture and meniscal damage and were randomly assigned to one of four treatment groups with varying doses of P188. ACL reconstruction was then performed using an autograft from the semitendinosus tendon. Animals were euthanized 1-month following trauma, meniscal tissue was assessed for changes in morphology, mechanical properties, and proteoglycan content. Femurs and tibias were scanned using microcomputed tomography to determine changes in bone quality, architecture, and osteophyte formation. The medial meniscus experienced more damage and a decrease in the instantaneous modulus regardless of treatment group, while P188 treatment tended to limit degenerative changes in the lateral meniscus. Both lateral and medial menisci had documented decreases in the equilibrium modulus and inconsistent changes in proteoglycan content. Minimal changes were documented in the tibias and femurs, with the only significant change being the formation of osteophytes in both bones regardless of treatment group. The data suggest that P188 was able to limit some degenerative changes in the meniscus associated with PTOA and may warrant future studies.

Assessment of changes in the meniscus and subchondral bone in a novel closed-joint impact and surgical reconstruction lapine model.

Despite reconstruction surgery to repair a torn anterior cruciate ligament (ACL), patients often still show signs of post-traumatic osteoarthritis (PTOA) years following the procedure. The goal of this study was to document changes in the meniscus and subchondral bone due to closed-joint impact and surgical reconstruction in a lapine model. Animals received insult to the joint followed by surgical reconstruction of the ACL and partial meniscectomy. Following euthanasia of the animals at 1, 3, and 6-months post-impact, meniscal tissue was assessed for changes in morphology, mechanical properties and proteoglycan content. Femurs and tibias were scanned via micro-computed tomography to determine changes in bone quality, morphometry, and formation of osteophytes. Both the lateral and medial menisci showed severe degradation and tearing at all-time points, with higher degree of degeneration being observed at 6-months. Decreases in both the instantaneous and equilibrium modulus were documented in both menisci. Minimal changes were found in bone quality and morphometry, with most change documented in the tibia. Bones from the reconstructed limbs showed large volumes of osteophyte formations, with an increase in volume over time. The initial changes that were representative of PTOA may have been limited to the meniscus, but at later time points consistent changes due to the disease were seen in both tissues. This study, which builds on a previous study by this laboratory, suggests that the addition of surgical reconstruction of the ACL to our model was not sufficient to prevent the development of PTOA.

Loosening of Transtibial Pullout Meniscal Root Repairs due to Simulated Rehabilitation Is Unrecoverable: A Biomechanical Study.

PURPOSE: To determine whether meniscal root repairs recover from displacement due to rehabilitative loading. METHODS: Transtibial pullout repairs of the posteromedial meniscal root were performed in 16 cadaveric ovine knees. Single- and double-tunnel repairs using the 2-simple suture technique were cyclically loaded in tension to 10,000 cycles, allowed to rest, and loaded in tension again. Paired differences in displacement with rest were recorded to evaluate recoverability. Displacement of repairs at cycles of interest was recorded, and the response of repairs to 10,000 cycles was assessed. RESULTS: All outcomes were not significantly different between the single- and double-tunnel techniques; therefore, the results were pooled. The difference in displacement between the first cycle and the first cycle after rest was 1.59 ± 0.69 mm. Repair displacement did not reach an equilibrium within 10,000 cycles and instead resulted in a steady increase in displacement of 0.05 ± 0.02 mm per additional 1,000 cycles. Sutures macroscopically began to cut out of the meniscus in both single- and double-tunnel repairs. CONCLUSIONS: This study showed that significant, unrecoverable loosening from rehabilitative loading occurred in single- and double-tunnel meniscal root repairs. Root repairs also gradually displaced with continued loading instead of reaching an equilibrium displacement after 10,000 cycles. This progressive, unrecoverable loosening needs to be studied further to better understand the resultant impact on knee mechanics. In addition, the quality and quantity of meniscal root repair healing at the time of rehabilitation should be studied to determine how susceptible patients are to repair loosening. CLINICAL RELEVANCE: Rehabilitative loading caused unrecoverable and progressive loosening of root repairs, showing the importance of healing before loading. Investigations on the effects of loosening on mechanics and the quality of repair healing at weight bearing are necessary to better understand the clinical implications.

RALA complexed α-TCP nanoparticle delivery to mesenchymal stem cells induces bone formation in tissue engineered constructs in vitro and in vivo.

A range of bone regeneration strategies, from growth factor delivery and/or mesenchymal stem cell (MSC) transplantation to endochondral tissue engineering, have been developed in recent years. Despite their tremendous promise, the clinical translation and future use of many of these strategies is being hampered by concerns such as off target effects associated with growth factor delivery. Therefore the overall objective of this study was to investigate the influence of alpha-tricalcium phosphate (α-TCP) nanoparticle delivery into MSCs using an amphipathic cell penetrating peptide RALA, on osteogenesis in vitro and both intramembranous and endochondral bone formation in vivo. RALA complexed α-TCP nanoparticle delivery to MSCs resulted in an increased expression of bone morphogenetic protein-2 (BMP-2) and an upregulation in a number of key osteogenic genes. When α-TCP stimulated MSCs were encapsulated into alginate hydrogels, enhanced mineralization of the engineered construct was observed over a 28 day culture period. Furthermore, the in vivo bone forming potential of RALA complexed α-TCP nanoparticle delivery to MSCs was found to be comparable to growth factor delivery. Recognizing the potential and limitations associated with endochondral bone tissue engineering strategies, we then sought to explore how α-TCP nanoparticle delivery to MSCs influences early mineralization of engineered cartilage templates in vitro and their subsequent ossification in vivo. Despite accelerating mineralization of engineered cartilage templates in vitro, RALA complexed α-TCP nanoparticle delivery did not enhance endochondral bone formation in vivo. Therefore the potential of RALA complexed α-TCP nanoparticle delivery appears to be as an alternative to growth factor delivery as a single stage strategy for promoting bone generation.

Direct versus indirect ACL femoral attachment fibres and their implications on ACL graft placement.

PURPOSE: To further elucidate the direct and indirect fibre insertion morphology within the human ACL femoral attachment using scanning electron microscopy and determine where in the footprint each fibre type predominates. The hypothesis was that direct fibre attachment would be found centrally in the insertion site, while indirect fibre attachment would be found posteriorly adjacent to the posterior articular cartilage. METHODS: Ten cadaveric knees were dissected to preserve and isolate the entirety of the femoral insertion of the ACL. Specimens were then prepared and evaluated with scanning electron microscopy to determine insertional fibre morphology and location. RESULTS: The entirety of the fan-like projection of the ACL attachment site lay posterior to the lateral intercondylar ridge. In all specimens, a four-phase architecture, consistent with previous descriptions of direct fibres, was found in the centre of the femoral attachment site. The posterior margin of the ACL attachment attached directly adjacent to the posterior articular cartilage with some fibres coursing into it. The posterior portion of the ACL insertion had a two-phase insertion, consistent with previous descriptions of indirect fibres. The transition from the ligament fibres to bone had less interdigitations, and the interdigitations were significantly smaller (p < 0.001) compared to the transition in the direct fibre area. The interdigitations of the direct fibres were 387 ± 81 µm (range 282-515 µm) wide, while the interdigitations of indirect fibres measured 228 ± 75 µm (range 89-331 µm). CONCLUSIONS: The centre of the ACL femoral attachment consisted of a direct fibre structure, while the posterior portion had an indirect fibre structure. These results support previous animal studies reporting that the centre of the ACL femoral insertion was comprised of the strongest reported fibre type. Clinically, the femoral ACL reconstruction tunnel should be oriented to cover the entirety of the central direct ACL fibres and may need to be customized based on graft type and the fixation device used during surgery.

A biomechanical evaluation of anterior and posterior tibialis tendons as suitable single-loop anterior cruciate ligament grafts.

PURPOSE: Because allograft tendons used to replace a torn anterior cruciate ligament are in short supply, it is useful to explore other possible graft sources. The purpose of this study was to determine whether a graft formed from a loop of either an anterior or posterior tibialis tendon has structural, material, and viscoelastic properties similar to those of a double-looped semitendinosus and gracilis (DLSTG) graft. TYPE OF STUDY: Completely randomized design. METHODS: Four structural and 3 material properties were determined for each type of graft (n = 10) by measuring the cross-sectional area, looping the tendon(s) over a post, gripping the free ends of the tendon(s) with a freeze clamp, and pulling the graft to failure by using a materials testing system. Two viscoelastic properties were determined for each type of graft (n = 10) by measuring the decrease in load under a constant displacement (i.e., stress relaxation test) and the increase in displacement under a constant load (i.e., creep test). RESULTS: For grafts 95 mm in length, the ultimate load and ultimate displacement of a loop of anterior (4,122 N, 12.0 mm) and posterior tibialis (3,594 N, 12.5 mm) tendon were either similar to or significantly greater than those of the DLSTG graft (2,913 N, 8.4 mm) (P =.204 for the posterior tibialis ultimate load and P < or =.007 for the remaining quantities). The stiffness and cross-sectional area of the anterior (460 N/mm, 48.2 mm2) and posterior tibialis (379 N/mm, 41.9 mm2) grafts were similar to those of the DLSTG graft (418 N/mm, 44.4 mm2) (P > or =.283). The tensile modulus, stress at ultimate load, and strain at ultimate load of the anterior tibialis and posterior tibialis grafts were either similar to or significantly greater than those of the DLSTG graft. The decrease in load of the anterior tibialis and posterior tibialis grafts was either greater than or similar to that of the DLSTG graft for the relaxation test (P < or =.066). The increase in displacement of the anterior tibialis (0.3 mm) and posterior tibialis (0.4 mm) grafts was minimally but significantly greater than that of the DLSTG graft (0.2 mm) for the creep test (P < or =.004). CONCLUSIONS: The structural, material, and viscoelastic properties of a single loop of anterior tibialis and posterior tibialis tendon are either better than or similar to those of a DLSTG graft. Consequently, single-loop grafts formed from tibialis tendons should function well as a replacement for a torn anterior cruciate ligament.