Background: Micronized dehydrated human amnion/chorion membrane (mdHACM) has reduced short term post-traumatic osteoarthritis (PTOA) progression in rats when delivered 24 h after medial meniscal transection (MMT) and is being investigated for clinical use as a disease modifying therapy. Much remains to be assessed, including its potential for longer-term therapeutic benefit and treatment effects after onset of joint degeneration.Objectives: Characterize longer-term effects of acute treatment with mdHACM and determine whether treatment administered to joints with established PTOA could slow or reverse degeneration. Hypotheses: Acute treatment effects will be sustained for 6 weeks, and delivery of mdHACM after onset of joint degeneration will attenuate structural osteoarthritic changes.Methods: Rats underwent MMT or sham surgery (left leg). mdHACM was delivered intra-articularly 24 h or 3 weeks post-surgery (n = 5–7 per group). Six weeks post-surgery, animals were euthanized and left tibiae scanned using equilibrium partitioning of an ionic contrast agent microcomputed tomography (EPIC-µCT) to structurally quantify joint degeneration. Histology was performed to examine tibial plateau cartilage.Results: Quantitative 3D µCT showed that cartilage structural metrics (thickness, X-ray attenuation, surface roughness, exposed bone area) for delayed mdHACM treatment limbs were significantly improved over saline treatment and not significantly different from shams. Subchondral bone mineral density and thickness for the delayed treatment group were significantly improved over acute treated, and subchondral bone thickness was not significantly different from sham. Marginal osteophyte degenerative changes were decreased with delayed mdHACM treatment compared to saline. Acute treatment (24 h post-surgery) did not reduce longer-term joint tissue degeneration compared to saline. Histology supported µCT findings and further revealed that while delayed treatment reduced cartilage damage, chondrocytes displayed qualitatively different morphologies and density compared to sham.Conclusion: This study provides insight into effects of intra-articular delivery timing relative to PTOA progression and the duration of therapeutic benefit of mdHACM. Results suggest that mdHACM injection into already osteoarthritic joints can improve joint health, but a single, acute mdHACM injection post-injury does not prevent long term osteoarthritis associated with meniscal instability. Further work is needed to fully characterize the durability of therapeutic benefit in stable osteoarthritic joints and the effects of repeated injections.

Osteoarthritis (OA) is a widespread disease that continues to lack approved and efficacious treatments that modify disease progression. Micronized dehydrated human amnion/chorion membrane (mu-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. The objectives of this study were to characterize the particle size distributions of two mu-dHACM formulations and to investigate the influence of these distributions on the in vivo therapeutic efficacy of mu-dHACM. Male Lewis rats underwent medial meniscus transection (MMT) or sham surgery, and intra-articular injections of saline, mu-dHACM, or reduced particle size mu-dHACM (RPS mu-dHACM) were administered at 24 hours postsurgery (n = 9 per treatment group). After 3 weeks, the animals were euthanized, and left legs harvested for equilibrium partitioning of an ionic contrast agent microcomputed tomography and histological analysis. mu-dHACM and RPS mu-dHACM particles were fluorescently tagged and particle clearance was tracked in vivo for up to 42 days postsurgery. Protein elution from both formulations was quantified in vitro. Treatment with mu-HACM, but not RPS mu-dHACM, reduced lesion volume in the MMT model 3 weeks postsurgery. In contrast, RPS mu-dHACM increased cartilage surface roughness and osteophyte cartilage thickness and volume compared to saline treatment. There was no difference of in vivo fluorescently tagged particle clearance between the two mu-dHACM sizes. RPS mu-dHACM showed significantly greater protein elution in vitro over 21 days. Overall, delivery of RPS mu-dHACM did result in an increase of in vivo joint degeneration and in vitro protein elution compared to mu-dHACM, but did not result in differences in joint clearance in vivo. These results suggest that particle size and factor elution may be tailorable factors that are important to optimize for particulate amniotic membrane treatment to be an effective therapy for OA. Impact Statement Osteoarthritis (OA) is a widespread disease that continues to lack treatments that modify the progression of the disease. Micronized dehydrated human amnion/chorion membrane (mu-dHACM) has been shown to be effective in reducing OA progression, but many of the engineering design parameters have not been explored. This work investigates the effects of particle size profile of the mu-dHACM particles and lays out the methods used in these studies. The results of this work will guide engineers in designing mu-dHACM treatments specifically and disease-modifying OA therapeutics generally, and it demonstrates the utility of novel therapeutic evaluation methods such as contrast-enhanced microcomputed tomography.

As a potential treatment for osteoarthritis (OA), we have developed injectable and hydrolytically degradable heparin-based biomaterials with tunable sulfation for the intra-articular delivery of tumor necrosis factor-alpha stimulated gene-6 (TSG-6), a protein known to inhibit plasmin which may degrade extracellular matrix within OA joints. We first assessed the effect of heparin sulfation on TSG-6 anti-plasmin activity and found that while fully sulfated (Hep) and heparin desulfated at only the N position (Hep-N) significantly enhanced TSG-6 bioactivity in vitro, fully desulfated heparin (Hep-) had no effect, indicating that heparin sulfation plays a significant role in modulating TSG-6 bioactivity. Next, TSG-6 loaded, degradable 10 wt% Hep-N microparticles (MPs) were delivered via intra-articular injection into the knee at 1, 7, and 15 days following medial meniscal transection (MMT) injury in a rat model. After 21 days, cartilage thickness, volume, and attenuation were significantly increased with soluble TSG-6, indicating degenerative changes. In contrast, no significant differences were observed with TSG-6 loaded MP treatment, demonstrating that TSG-6 loaded MPs reduced cartilage damage following MMT injury. Ultimately, our results indicate that Hep-N can enhance TSG-6 anti-plasmin activity and that Hep-N-based biomaterials may be an effective method for TSG-6 delivery to treat OA.

PURPOSE: Either reduced or elevated joint loading has been associated with post-traumatic osteoarthritis (OA); however, which altered loading condition may be more detrimental to cartilage health post-injury remains unknown. This study examined the effects of reduced and elevated joint loading on cartilage degeneration, knee locomotion kinematics, and degree of voluntary activity in rats following medial meniscal transection (MMT). METHODS: A total of 22 male Lewis rats (weight: 304 ± 57 gm) underwent MMT in their left hind-limbs and were assigned to one of the three conditions: 1) regular loading (N = 7), 2) reduced loading via hind-limb immobilization (N = 8), or 3) elevated loading via daily treadmill running (N = 7). A sham surgery was performed in 7 separate rats. Rats were evaluated pre-MMT and 8 weeks post-MMT for the amount of voluntary daily run time/distance on a running wheel and hind-limb joint kinematics during treadmill locomotion (speed: 30 m/min) using a 3D X-ray motion analysis. Rats were euthanized after 8 weeks and the 3D microstructure and composition of the tibial plateau cartilage and subchondral bone was quantified using contrast-enhanced microcomputed tomography. RESULTS: When compared to the elevated-loading group at the 8th week post-MMT, the reduced-loading group demonstrated a greater reduction in voluntary run time (47.7 ± 46.8 % vs. 18.0 ± 69.9 %, P = 0.043) and distance (57.2 ± 38.3 % vs. 19.7 ± 81.2 %, P = 0.029). Cartilage data from 4 rats per group indicated that the elevated-loading rats had the greatest lesion/exposed bone area and subchondral bone volume (0.50 ± 0.35 um2 and 1.16 ± 0.24 mm3, respectively), followed by the regular-loading rats (0.43 ± 0.19 um2 and 1.06 ± 0.22 mm3) and reduced-loading rats (0.14 ± 0.17 um2 and 0.97 ± 0.03 mm3). All three MMT groups demonstrated a more extended knee position (by about 8-18°) at mid-stance during locomotion when compared to the sham rats. CONCLUSIONS: Our current findings suggest that while elevating joint loading (via treadmill running) exacerbated post-traumatic OA, reducing joint loading (via joint immobilization) may delay OA progression in MMT rats. However, the difference in cartilage degeneration among different loading conditions may not correlate with the behavior changes in voluntary activity and knee locomotion kinematics.