(227d) Amnion Pillow for Osteoarthritis: A Novel Regenerative Approach

Background and Purpose: Osteoarthritis (OA), the most common degenerative joint disease, affects over 32 million adults in the United States and more than 500 million people globally, representing approximately 7.6% of the population. OA is characterized by progressive cartilage degradation, synovial inflammation, and subchondral bone remodeling, causing chronic pain, joint stiffness, and significant mobility impairments, particularly in aging populations and those with risk factors such as obesity and joint trauma. The socioeconomic burden of OA is substantial, with annual healthcare costs exceeding $185 billion in the U.S. alone, driven by medical interventions and productivity losses.

Current treatments, including analgesics, nonsteroidal anti-inflammatory drugs, intra-articular corticosteroid or hyaluronic acid injections, and physical therapy, focus on symptom management but fail to address the underlying pathological mechanisms of cartilage loss and inflammation. This therapeutic gap underscores the urgent need for regenerative strategies that can halt disease progression and restore joint function. Human amniotic membrane (AM), derived from perinatal placental tissue, offers a promising solution due to its anti-inflammatory, immunomodulatory, and chondroprotective properties. AM suppresses proinflammatory cytokines (e.g., IL-1β, TNF-α), releases growth factors to promote tissue repair, and recruits stem cells to enhance cartilage regeneration, making it an ideal candidate for OA therapy.

This study evaluates two AM-based interventions: decellularized AM patches and a novel "amnion pillow" designed to mechanically cushion knee joints, reduce cartilage stress, and mitigate inflammation, aiming to bridge the gap between symptomatic relief and disease modification. The amnion pillow concept builds upon these biological strengths while introducing key innovations to enhance therapeutic efficacy and translational potential. By engineering the amnion into a compact, pillow-like configuration, this platform increases the payload of bioactive components, enables controlled and sustained factor release, and provides a mechanical cushioning effect within the joint space. Moreover, the pillow structure offers versatility for incorporating additional therapeutic agents, supporting a multimodal, patient-specific treatment strategy. Importantly, the use of amnion biomaterials enhances the feasibility of regulatory translation and clinical application. This strategy has the potential to shift the current OA treatment paradigm by offering a single therapeutic that addresses both symptomatic relief and disease modification.

Methods:

1. AM Decellularization and DNA Quantification: Human amnion is a rich source of extracellular matrix (ECM) proteins, cytokines, and growth factors. However, improper decellularization of tissue samples can lead to immune rejection. Amnion decellularization with sodium hydroxide (NaOH, 0.5 M) was employed. Under sterile conditions, harvested amnion was washed with normal saline containing 100 units/mL penicillin and 100 µg/mL streptomycin to remove blood and mucus. The membrane was decellularized by dipping and scraping with gauze pads in 0.5 M NaOH for 1 minute, followed by rinsing in copious amounts of deionized water five times or until all NaOH, which is toxic to cells, was completely eliminated. Decellularization was validated by nuclear staining with propidium iodide to assess removal of nuclear content under a microscope and by DNA quantification using a PicoGreen assay kit (ELISA) to measure the residual DNA content in the tissue after processing. Glycosaminoglycan (GAG) and collagen quantifications were performed to confirm retention of bioactive components, such as growth factors.
2. Development of Amnion Pillow: The process for creating the amnion pillow involves careful handling of the decellularized amnion membrane to ensure it retains its biological properties while being transformed into a functional therapeutic device. Glutaraldehyde was used as the cross-linker of choice for the study due to its established role in cross-linking collagen and other ECM components. To minimize toxicity, glutaraldehyde was used in the smallest effective quantity to avoid excessive cross-linking, which can compromise the membrane’s biocompatibility. The amnion pillow prototype was developed by dipping forceps into a 10% glutaraldehyde solution (in water), grasping one edge of the amnion membrane, and holding it for 5 minutes. With the pillow oriented upward, gravity minimized glutaraldehyde absorption by the membrane. This sealing process was repeated for the other two sides, creating a fully sealed amnion pillow. This approach ensures that the membrane retains its integrity and biological properties while forming the desired pillow-like shape. The pillow was thoroughly rinsed with deionized water to remove residual glutaraldehyde and then freeze-dried for later use.
3. Degradation: Amnion pillow degradation studies were conducted in 30 U/mL collagenase type II in phosphate-buffered saline (PBS) at 37°C, with corresponding weight loss measured for each sample.
4. Evaluation of Biocompatibility and Anti-inflammatory Effects of Amnion Pillow: Rat chondrocytes were plated into 24-well plates at a density of 1×10^5 cells/well using DMEM-F12 with 10% FBS and 1% penicillin/streptomycin and cultured for 24 hours. The culture medium was then refreshed with DMEM-F12 containing 20 ng/mL IL-1β to induce chondrocyte inflammatory responses for an additional 24 hours. IL-1β-treated chondrocytes were co-cultured with transwell inserts containing one amnion Chondrocytes without IL-1β served as a negative control as well as to assess biocompatibility. The chondrocytes were tested for viability and nitric oxide (NO) production using MTS and NO assay kits, respectively.
5. In Vivo Experiments: Male Sprague-Dawley rats were used for the study. OA was induced in their knees by two injections of 500 U collagenase type II on days 0 and 3. The sham group received 100 μL of normal saline. One week after the first collagenase injection, experimental groups received amniotic grafts through open surgery. Knee diameters were measured to determine the extent of joint swelling using a manual caliper. Six weeks after implantation of amniotic grafts, rats were sacrificed, and dissected knees were used for hematoxylin and eosin (H&E) staining.

Results:

1. Propidium iodide (PI) staining of decellularized amnion patches showed minimal residual nuclear content under a microscope, and the PicoGreen assay revealed residual DNA content of less than 50 ng per 1 mg dry weight of the tissue, which is within acceptable limits.
2. In vitro studies showed that AM significantly reduced nitric oxide concentration in the culture medium, indicating reduced inflammation due to the tissue’s anti-inflammatory properties. Moreover, AM exhibited no toxicity and promoted chondrocyte proliferation.
3. Amnion pillow enzymatic degradation studies, conducted in 30 U/mL collagenase type II in PBS at 37°C over 8 days, showed approximately 45% degradation by day 8.
4. Joint swelling measurements showed significant reductions and decreased inflammation after AM implantation. Histological staining revealed that AM-implanted groups had reduced fibrillation and erosion, smoother cartilage surfaces, and less inflammatory cell infiltration in the synovial membrane.

Conclusion:
The amnion pillow demonstrated excellent biocompatibility and minimal swelling, degradation, or toxicity, with high cell viability, confirming its clinical potential. In vitro, AM pillows significantly reduced proinflammatory markers in inflamed chondrocytes, showcasing robust anti-inflammatory effects. In vivo, AM implantation in a rat OA model significantly reduced joint swelling over six weeks compared to controls, though histological joint scores showed no notable differences. AM-based interventions, including the innovative amnion pillow, show significant promise for alleviating OA-associated inflammation and cartilage damage. Longer-term studies may clarify chondroprotective effects. These findings support AM-based therapies as minimally invasive, regenerative solutions for OA, with potential for clinical translation.