Effect of amniotic suspension allograft in a rat destabilization of medial meniscus osteoarthritis model.

Placental-derived allografts have been of interest as a potential nonsurgical treatment to reduce pain and improve function in knee osteoarthritis (OA). The purpose of this study was to evaluate the effect of single and repeat injection of amniotic suspension allograft (ASA) on pain, function, and cytokine levels using a destabilization of the medial meniscus (DMM) rat model of OA. Post-DMM surgery, animals were treated with a single injection of either ASA, vehicle, or triamcinolone, or repeated injection of either ASA or vehicle. Behavioral testing including knee swelling, pain threshold, dynamic weight bearing (DWB), and gait analysis were evaluated during the in-life phase. Postsacrifice, histopathology and serum and synovial fluid analyses were evaluated. Significant improvements in both DWB differentials and pain threshold were seen in response to repeated injection of ASA, while a single injection of ASA and triamcinolone resulted in significant improvements in pain threshold. Histopathology analysis found no significant differences regardless of treatment compared to vehicle, except for an increase in synovitis following repeated injection of ASA. A single injection of ASA and triamcinolone resulted in increased anti-inflammatory cytokines; repeated ASA injection resulted in significant increases in several immune-modulating factors relevant to OA. When comparing the impact of single and repeat ASA treatments on behavioral testing, repeated injection provided significant additional improvements in both pain and function. This study provides evidence demonstrating the impact of a second injection while also providing additional data for evaluating the use of ASA as a nonsurgical treatment for knee OA.

Retention of Key Characteristics of Unprocessed Chorion Tissue Resulting in a Robust Scaffold to Support Wound Healing.

Placental membranes have been widely studied and used clinically for wound care applications, but there is limited published information on the benefits of using the chorion membrane. The chorion membrane represents a promising source of placental-derived tissue to support wound healing, with its native composition of extracellular matrix (ECM) proteins and key regulatory proteins. This study examined the impact of hypothermic storage on the structure of chorion membrane, ECM content, and response to degradation in vitro. Hypothermically stored chorion membrane (HSCM) was further characterized for its proteomic content, and for its functionality as a scaffold for cell attachment and proliferation in vitro. HSCM retained the native ECM structure, composition, and integrity of native unprocessed chorion membrane and showed no differences in response to degradation in an in vitro wound model. HSCM retained key regulatory proteins previously shown to be present in placental membranes and promoted the attachment and proliferation of fibroblasts in vitro. These data support the fact that hypothermic storage does not significantly impact the structure and characteristics of the chorion membrane compared to unprocessed tissue or its functionality as a scaffold to support tissue growth.

An Initial Injection and a Crossover Injection of Amniotic Suspension Allograft Following Failed Treatment with Hyaluronic Acid or Saline Are Equally Effective in the Treatment of Moderate Symptomatic Knee Osteoarthritis Over 12 Months.

PURPOSE: The purpose of this crossover study was to determine the efficacy of amniotic suspension allograft (ASA) for moderate symptomatic knee osteoarthritis following failed treatment with hyaluronic acid (HA) or saline through 12 months' postcrossover injection using patient-reported and safety outcomes. METHODS: In this multicenter study, 95 patients from a 200-patient single-blind randomized controlled trial were eligible to crossover and receive a single injection of ASA 3 months after failed treatment with HA or saline. Patient-reported outcomes, including Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS), were collected out to 12 months postcrossover to determine pain and function. Radiographs and blood were collected for assessment of changes. Statistical analyses were performed using mixed effects model for repeated measures. RESULTS: Treatment with ASA following failed treatment with HA or saline resulted in significant improvements in KOOS and VAS scores compared with crossover baseline. There were no differences in radiographic measures or anti-human leukocyte antigen serum levels compared with baseline and no severe adverse events reported. In addition, more than 55% of patients were responders at months 3, 6, and 12 as measured by the Outcome Measures in Arthritis Clinical Trials-Osteoarthritis Research Society International simplified responder criteria. There were no significant differences between the original ASA randomized group and crossover cohorts at any of the time points evaluated, suggesting that prior failed treatment with HA or saline did not significantly impact outcomes following treatment with ASA. CONCLUSIONS: This study showed that patients who previously failed treatment with HA or saline had statistically significant improvements in pain and function scores following a crossover injection of ASA that was sustained for 12 months, as measured by KOOS and VAS. There were no serious adverse events reported, and the injection was safe. LEVEL OF EVIDENCE: II, prospective cohort study.

Bi-layered living cellular construct resulted in greater healing in an alloxan-induced diabetic porcine model.

Tissue-engineered skin constructs, including bi-layered living cellular constructs (BLCC) used in the treatment of chronic wounds, are structurally/functionally complex. While some work has been performed to understand their mechanisms, the totality of how BLCC may function in wound healing remains unknown. To this end, we have developed a delayed wound healing model to test BLCC cellular and molecular mechanisms of action. Diabetes was chemically-induced using alloxan in Yucatan miniature pigs, and full-thickness wounds were generated on their dorsum. These wounds were either allowed to heal by secondary intention alone (control) or treated with a single or multiple treatments of a porcine autologous BLCC. Results indicated a single treatment with porcine BLCC resulted in statistically significant wound healing at day 17, while four treatments resulted in statistically significant healing on days 10, 13, and 17 compared to control. Statistically accelerated wound closure was driven by re-epithelialisation rather than contraction or granulation. This porcine diabetic model and the use of a porcine BLCC allowed evaluation of healing responses in vivo without the complications typically seen with either xenogenic responses of human/animal systems or the use of immune compromised animals, expanding the knowledge base around how BLCC may impact chronic wounds.

Amniotic suspension allograft improves pain and function in a rat meniscal tear-induced osteoarthritis model.

BACKGROUND: Osteoarthritis is a degenerative disease of the knee that affects 250 million people worldwide. Due to the rising incidence of knee replacement and revision surgery, there is a need for a nonsurgical treatment to reduce pain and improve function in patients with knee osteoarthritis. Placental-derived allografts, such as an amniotic suspension allograft (ASA), provide growth factors and cytokines that could potentially modulate the inflammatory environment of osteoarthritis. The purpose of this study was to evaluate the efficacy of ASA in a rat medial meniscal tear (MMT) induced osteoarthritis model through histology, microCT, synovial fluid biomarkers, and behavioral testing. METHODS: Rats underwent MMT surgery at day - 7; at day 0, rats were injected with either ASA, vehicle control, or fibroblast growth factor-18 (FGF18). Behavioral testing, including gait analysis, pain threshold, incapacitance, and knee swelling were evaluated in-life, along with histology, microCT analysis of cartilage, and synovial fluid testing post-sacrifice. One MMT cohort was sacrificed at day 10, the other at day 21. A third cohort acted as a safety arm and did not receive MMT surgery; these rats were injected with either vehicle control or ASA and evaluated at day 3 and day 21. RESULTS: Behavioral testing showed a significant improvement in pain threshold, incapacitance, and gait following an injection of ASA. MicroCT showed significant improvements in cartilage thickness and attenuation at day 10 only, and histology showed no detrimental effects compared to the vehicle control at day 21. Synovial fluid analysis showed a significant increase in anti-inflammatory IL-10. The safety cohort showed no significant differences except for an increase in synovitis at day 21, which could be evidence of a xenogeneic response in this model. CONCLUSIONS: In this study, an injection of ASA was well tolerated with no adverse events. Improvements in pain and function, along with cartilage properties at day 10, were observed. Increases in anti-inflammatory cytokines was also seen, along with no significant cartilage degeneration at day 21 compared to the vehicle control. This study provides evidence for the use of ASA as a nonsurgical treatment for knee OA.

Hypothermically Stored Amniotic Membrane for the Treatment of Cartilage Lesions: A Single-Arm Prospective Study with 2-Year Follow-Up.

OBJECTIVE: The purpose of this study was to determine the safety and efficacy of hypothermically stored amniotic membrane (HSAM) for the treatment of cartilage lesions of the knee using imaging, patient-reported outcomes (PROs), second-look arthroscopy, and histology. Patients were treated with HSAM and followed for 2 years. DESIGN: Subjects with focal chondral lesions of the femur (International Cartilage Repair Society grade 3-4) were enrolled in this single-arm prospective study. Standard of care imaging was completed. PROs, including the Knee Injury and Osteoarthritis Outcome Score (KOOS), Marx Activity Scale, and Visual Analog Scale (VAS), were collected at baseline and at 3, 6, 12, 18, and 24 months. Three subjects underwent an optional arthroscopy and biopsy of the repair at 24 months. RESULTS: Ten subjects were enrolled and completed the study. At 24 months, KOOS Sports & Recreation improved 173.3% and Quality of Life improved 195.3% from baseline. Marx Activity Scale increased 266.8% from 12 to 24 months. VAS scores improved 84.8% and 81.0% from baseline to 24 months for average and maximum pain. Modified Magnetic Resonance Observation of Cartilage Repair Tissue (MOCART) scoring showed that 7 of 10 subjects had complete defect repair and filling by 24 months. Biopsy staining for collagen II revealed integration and remodeling of HSAM into a mix of hyaline-like cartilage and fibrocartilage matrix. CONCLUSION: This study provides evidence supporting the safety and efficacy of HSAM for treating symptomatic cartilage lesions. Subjects showed a high degree of defect fill and integration with the native cartilage and reported improvements in pain and function post-treatment. Results provide important original data for future clinical trials.

A prospective clinical trial evaluating changes in the wound microenvironment in patients with chronic venous leg ulcers treated with a hypothermically stored amniotic membrane.

Amniotic tissues have been long utilised to treat chronic wounds; however, there are few studies evaluating how the wound microenvironment responds to these therapies. The goal of this study was to evaluate the changes in wounds treated with a hypothermically stored amniotic membrane (HSAM). In this prospective single-arm study, 15 female patients with venous leg ulcers were treated with HSAM from male donors and standard of care for 12 weeks. Over the course of the study, wound exudate was collected and evaluated using proteomic microarrays. Biopsies were collected during the course of treatment to detect the presence of HSAM tissue. By 4 weeks, 60% of subjects achieved 50% or greater reduction in wound size, and by 12 weeks, 53% of subjects achieved 100% re-epithelialization. HSAM DNA was detected in 20% of biopsies as determined by the detection TSPY4, indicating HSAM was no longer present within the wound bed approximately 7 days from the last treatment for the majority of wounds. Proteomic analysis of wound exudate found that wounds on a healing trajectory had significantly higher levels of MMP-10, MMP-7, and TIMP-4 and significantly lower levels of CX3CL1, FLT-3 L, IL-1ra, IL-1a, IL-9, IL-2, IL-3, MCP-1, and TNF-b compared with other wounds.

Safety and Efficacy of an Amniotic Suspension Allograft Injection Over 12 Months in a Single-Blinded, Randomized Controlled Trial for Symptomatic Osteoarthritis of the Knee.

PURPOSE: The purpose of this study is to determine the efficacy of amniotic suspension allograft (ASA) compared to hyaluronic acid (HA) and saline at up to 12 months of follow-up through the use of patient-reported outcomes, immunoglobulin levels, and anti-human leukocyte antigen (HLA) levels. METHODS: Within this multicenter study, 200 patients were randomized 1:1:1 to a single intra-articular injection of saline, HA, or ASA. Patient-reported outcomes, including Knee Injury and Osteoarthritis Outcome Score (KOOS) and visual analog scale (VAS) score, were collected at multiple time points (baseline, 1 week, 6 weeks, 3 months, 6 months) out to 12 months to assess improvements in pain and function. Radiographs at baseline and 12 months were taken to determine radiographic changes, while blood was collected at baseline, 6 weeks, and 6 months to determine changes in immunoglobulins and anti-HLA levels. Statistical analyses were performed using last observation carried forward and mixed effects model for repeated measures. RESULTS: Treatment with ASA resulted in significant improvements in KOOS and VAS scores that were maintained through 12 months (P < .05). Treatment with ASA resulted in a 63.2% responder rate at 12 months using the Outcome Measures in Arthritis Clinical Trials-Osteoarthritis Research Society International simplified definition. There were no significant differences between groups for radiographic measures in the index knee, immunoglobulins, C-reactive protein, or anti-HLA serum levels (P > .05). The number and type of adverse events (AEs) reported for ASA were comparable to the HA injection group, while no treatment-emergent AEs were reported for the saline group. CONCLUSIONS: This randomized controlled trial of ASA vs HA and saline for the treatment of symptomatic knee osteoarthritis demonstrated clinically meaningful improved outcomes with ASA over the controls out to 12 months postinjection. No concerning immunologic or adverse reactions to the ASA injection were identified with regards to severe AEs, immunoglobulin, or anti-HLA levels. LEVEL OF EVIDENCE: Level I, randomized controlled multicenter trial.

Amniotic Suspension Allograft Modulates Inflammation in a Rat Pain Model of Osteoarthritis.

Osteoarthritis (OA) affects over 301 million adults worldwide. Inflammation is a recognized component of the OA process; two potent pro-inflammatory cytokines involved in OA are interleukin-1ß and tumor necrosis factor-α. Placental-derived tissues and fluids are known to contain anti-inflammatory and immunomodulatory cytokines and growth factors. The objective of this study was to evaluate the anti-inflammatory effects of amniotic suspension allograft (ASA) in an in vivo model of OA; we evaluated pain, function, and cytokine levels following ASA treatment in the rat monosodium iodoacetate (MIA) OA pain model. Rats were injected with 2 mg of MIA, which causes pain, cartilage degeneration, and inflammation, followed by treatment with saline, triamcinolone (positive control), or ASA 7 days following disease induction with MIA. Behavioral assays, including gait analysis, mechanical pain threshold, incapacitance, and swelling were evaluated, along with histology and serum and synovial fluid biomarkers. Treatment with ASA resulted in significant improvements in pain threshold, while weight bearing aversion and swelling were significantly decreased. There were no differences between groups in total joint score after histological grading. Serum biomarkers did not show differences, indicating a lack of systemic response; however, synovial fluid levels of IL-10 were significantly increased in animals treated with ASA. ASA treatment significantly reduced pain, weight-bearing aversion and swelling. This study provides mechanistic data regarding potential therapeutic effects of ASA in OA and preliminary evidence of the anti-inflammatory nature of ASA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 38:1141-1149, 2020.

Treatment with Human Amniotic Suspension Allograft Improves Tendon Healing in a Rat Model of Collagenase-Induced Tendinopathy.

Treatment of tendon injuries is challenging, with neither conservative nor surgical approaches providing full recovery. Placental-derived tissues represent a promising tool for the treatment of tendon injuries. In this study, human amniotic suspension allograft (ASA) was investigated in a pre-clinical model of Achilles tendinopathy. Collagenase type I was injected in the right hind limb of Sprague Dawley rats to induce disease. Contralateral tendons were either left untreated or injected with saline as controls. Seven days following induction, tendons were injected with saline, ASA, or left untreated. Rats were sacrificed 14 and 28 days post-treatment. Histological and biomechanical analysis of tendons was completed. Fourteen days after ASA injection, improved fiber alignment and reduced cell density demonstrated improvement in degenerated tendons. Twenty-eight days post-treatment, tendons in all treatment groups showed fewer signs of degeneration, which is consistent with normal tendon healing. No statistically significant differences in histological or biomechanical analyses were observed between treatment groups at 28 days independent of the treatment they received. In this study, ASA treatment was safe, well-tolerated, and resulted in a widespread improvement of the tissue. The results of this study provide preliminary insights regarding the potential use of ASA for the treatment of Achilles tendinopathy.

A Randomized Controlled Single-Blind Study Demonstrating Superiority of Amniotic Suspension Allograft Injection Over Hyaluronic Acid and Saline Control for Modification of Knee Osteoarthritis Symptoms.

Placental-derived tissues are a known source of anti-inflammatory and immune modulating factors. Published pilot data on amniotic suspension allograft (ASA) for the treatment of osteoarthritis (OA) demonstrated safety and trends for improved pain and function. A multicenter randomized controlled trial was designed to evaluate the efficacy of symptom modulation with ASA compared with saline and hyaluronic acid (HA) in subjects with knee OA. A total of 200 subjects were randomized 1:1:1 to ASA, HA, or saline, with subjects blinded to their allocation. Changes from baseline of patient-reported outcomes (PROs)-EQ-5D-5L, Knee Osteoarthritis Outcome Score (KOOS), visual analog scale (VAS), Tegner, and Single Assessment Numerical Evaluation (SANE)-were compared between groups. Patients reporting unacceptable pain at 3 months were considered treatment failures and withdrawn from the study. Statistical analysis was completed by comparing changes in PROs from baseline to 3 and 6 months for all groups. Comparison of demographics between treatment groups showed no significant differences between groups. Patients reporting unacceptable pain at 3 months in each group were ASA (13.2%), HA (68.8%), and saline (75%). Patients receiving ASA demonstrated significantly greater improvements from baseline for overall pain (VAS), KOOS pain, and KOOS-activities of daily living scores compared with those in the HA group (3 months) and both groups (6 months). ASA patients had significantly greater improvements in KOOS symptom scores compared with HA and saline at 3 and 6 months, respectively. OMERACT-OARSI responder rates for ASA, HA, and saline groups were 69.1, 39.1, and 42.6%, respectively (p = 0.0007). Subjects receiving ASA treatment showed greater improvements in PROs and fewer patients reported unacceptable pain compared with HA and saline. The evidence presented in this Level I Randomized Controlled Trial suggests that ASA injection is an effective treatment for the nonoperative management of symptomatic knee OA.

A mechanistic evaluation of the angiogenic properties of a dehydrated amnion chorion membrane in vitro and in vivo.

Angiogenesis is essential for the successful repair of tissues; however, in many chronic conditions, angiogenesis is inhibited. Placental tissues have been shown to illicit an angiogenic response both in vitro and in vivo, and the angiogenic properties of these tissues likely contribute to observed clinical outcomes. Although there is some work describing the angiogenic effects of these tissues, comparatively little has been done to determine the possible mechanisms responsible for this effect. The purpose of this study was to conduct a thorough evaluation of a commercially available dehydrated amnion chorion membrane to better understand how these tissues may promote angiogenesis. The proteomic content of this tissue was evaluated using a high throughput proteomic microarray, and then the effects of these grafts were evaluated in vivo using subcutaneous gelfoam sponge implants containing conditioned media (CM) from the graft. Human microvascular endothelial cells were then used to determine how released factors effect migration, proliferation, gene expression, and protein production in vitro. Finally, to elucidate potential signaling-pathways through which tissue-derived factors act to induce pro-angiogenetic phenotypes in endothelial cells in vitro, we performed a global analysis of both serine/threonine and tyrosine kinase activity. Kinomic and proteomic data were then combined to generate protein-protein interaction networks that enabled the identification of multiple growth factors and cytokines with both pro- and anti-angiogenetic properties. In vivo, the addition of CM resulted in increased CD31 and αSMA staining and increases in pro-angiogenic gene expression. In vitro, CM resulted in significant increases in endothelial proliferation, migration, and the expression of granulocyte-macrophage colony-stimulating factor, hepatocyte growth factor, and transforming growth factor beta-3. Integrated kinomic analysis implicated ERK1/2 signaling as the primary pathway activated following culture of endothelial cells with dehydrated amnion/chorion membrane (dACM) CM. In conclusion, dACM grafts triggered pro-angiogenic responses both in vitro and in vivo that are likely at least partially mediated by ERK1/2 signaling.

Evaluation of two distinct placental-derived membranes and their effect on tenocyte responses in vitro.

Tendon healing is a complex, multiphase process that results in increased scar tissue formation, leading to weaker tendons. The purpose of this study was to evaluate the response of tenocytes to both hypothermically stored amniotic membrane (HSAM) and dehydrated amnion/chorion membrane (dACM). Composition and growth factor release from HSAM and dACM were evaluated using proteomics microarrays. HSAM and dACM releasate was used to assess tenocyte proliferation, migration, gene expression, extracellular matrix (ECM) protein deposition, and response to inflammation. Additionally, tenocyte-ECM interactions were evaluated. HSAM and dACM contain and release growth factors relevant to tendon healing, including insulin-like growth factor I, platelet-derived growth factor, and basic fibroblast growth factor. Both dACM and HSAM promoted increased tenocyte proliferation and migration; tenocytes treated with dACM proliferated more robustly, whereas treatment with HSAM resulted in higher migration. Both dACM and HSAM resulted in altered ECM gene expression; dACM grafts alone resulted in increases in collagen deposition. Furthermore, both allografts resulted in altered tenocyte responses to inflammation with reduced transforming growth factor beta levels. Additionally, dACM treatment resulted in increased expression and production of matrix metalloprotease-1 (MMP-1), whereas HSAM treatment resulted in decreased production of MMP-1. Tenocytes migrated into and remodeled HSAM only. These results indicate that both grafts have properties that support tendon healing; however, the results presented here suggest that the responses to each type of graft may be different. Due to the complex environment during tendon repair, additional work is needed to evaluate these effects using in vivo models.

Characterisation of dehydrated amnion chorion membranes and evaluation of fibroblast and keratinocyte responses in vitro.

The purpose of this study is to characterise the composition of a dehydrated amnion and chorion graft and investigate how factors released from this graft interact with cells important to the wound microenvironment using in vitro models. Characterisation was completed by proteomic analysis of growth factors and cytokines, evaluation of matrix components and protease inhibition, immunohistochemistry, and in vitro release of key growth factors and cytokines. To evaluate the effect of released factors on cells found within the microenvironment, in vitro assays including: cell proliferation, migration, gene expression, protein production, and intracellular pathway activation were used; additionally, responses of fibroblasts in the context of inflammation were measured. We found that released factors from dehydrated amnion/chorion membranes (dACM) stimulated cell proliferation, migration, and altered gene and protein expression profiles of cells important for wound repair in vitro. When cells were cultured in the presence of pro-inflammatory cytokines, the addition of releasate from dACM resulted in an altered production of cytokines, including a reduction of pro-inflammatory regulated on activation, normal T cell expressed and secreted (RANTES). In sum, the results presented here characterise the components of dACM, and in vitro studies were used to evaluate interactions of dACM with cell types important in wound healing.

Dehydrated Amnion/Chorion Improves Achilles Tendon Repair in a Diabetic Animal Model.

INTRODUCTION: Healing of tendon injuries is often plagued by significant scar formation and compromised biomechanical function. For those with diabetes, these injuries are further complicated by alterations to the extracellular matrix of the tendon, poor circulation, and delayed wound healing; consequently, complications and re-rupture rates for patients with diabetes are reported higher than the typical patient population. Placental derived membranes, specifically dehydrated human amnion/chorion membranes (dACMs), have been utilized clinically as an adhesion barrier, and these membranes have been shown to reduce scarring and aid in tissue repair. OBJECTIVE: The purpose of this study was to evaluate the effect of dACMs on tendon repair in a diabetic model with impaired healing. MATERIALS AND METHODS: Using a type II diabetic model (BBZDR/WOR rats), a full-thickness injury was made through the Achilles tendon and repaired using a modified Kessler method. Repaired tendons were wrapped with dACM or left unwrapped as a control (n = 15/group; n = 30 total). Tendons were retrieved at 14 (n = 5/group; n = 10 total) or 28 days (n = 10/group; n = 20 total) and evaluated using histology, immunofluorescence, and biomechanical testing. RESULTS: Treatment of tendons with dACM resulted in reduced failure rates, increased cell migration, and improved mechanical properties (compared with unwrapped controls). The dACM-treated tendons also showed changes in the production of several important biomarkers to tendon healing at both 14 and 28 days; most notably, Scleraxis was found to be upregulated in dACM-treated tendons. CONCLUSIONS: This study highlights a promising treatment option for this challenging clinical population.

Tenocyte cell density, migration, and extracellular matrix deposition with amniotic suspension allograft.

Amniotic suspension allografts (ASA), derived from placental tissues, contain particulated amniotic membrane and amniotic fluid cells. Recently, ASA and other placental-derived allografts have been used in orthopaedic applications, including tendinopathies and tendon injuries. The purpose of this study was to determine the potential effects of ASA on tenocyte cell density, migration, and responses to inflammatory stimuli. Tenocyte cell density was measured using AlamarBlue over multiple time points, while migration was determined using a Boyden chamber assay. Deposition of ECM markers were measured using BioColor kits. Gene expression and protein production of cytokines and growth factors following stimulus with pro-inflammatory IL-1ß and TNF-α was measured using qPCR and ELISAs. Conditioned media (CM) was made from ASA and used for all assays in this study. In vitro, ASA CM treatment significantly promoted tenocyte increases in cell density and migration compared to assay media controls. ASA CM also increased the deposition of extracellular matrix (ECM) proteins, including collagen, elastin, and sGAG. Following inflammatory stimulation and treatment with ASA CM, tenocytes downregulated IL-8 gene expression, a pro-inflammatory cytokine normally elevated during the inflammatory phase of tendon healing. Additionally, tenocytes treated with ASA CM had significantly lower protein levels of TGF-ß1 compared to controls. This study evaluated ASA and its effect on tenocytes; specifically, treatment with ASA resulted in increased cell density, more robust migration and matrix deposition, and some alteration of inflammatory targets. © 2018 The Authors. Journal of Orthopaedic Research® Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 37:412-420, 2019.

Evaluation of Percutaneous Intradiscal Amniotic Suspension Allograft in a Rabbit Model of Intervertebral Disc Degeneration.

STUDY DESIGN: A laboratory study using a rabbit annular puncture model of intervertebral disc degeneration (IDD). OBJECTIVE: The aims of this study were to assess whether an amniotic suspension allograft (ASA) containing particulated human amnion and amniotic fluid derived cells regains intervertebral disc height and morphology and improves histologic scoring in a rabbit model of IDD. SUMMARY OF BACKGROUND DATA: In contrast to current surgical interventions for IDD, in which the primary goal is to relieve symptomatic pain, one novel strategy involves the direct injection of anabolic cytokines. Current therapies for IDD are limited by both the short half-life of therapeutic proteins and general decline in anabolic cell populations. METHODS: Intervertebral discs in New Zealand white rabbits were punctured using 18-gauge needle under fluoroscopic guidance. Four weeks post-puncture, two groups of rabbits were injected with either ASA or a vehicle/sham control, while a third group was untreated. Weekly radiographs were obtained for 12 weeks to assess disc height index (DHI). Magnetic resonance imaging (MRI) T2 relaxation time was evaluated at weeks 4 and 12 to assess morphological changes. Histologic sections were evaluated on a semi-quantitative grading scale. RESULTS: Before treatment at week 4, DHIs and normalized T2 relaxation times between the three groups were not significantly different. At week 12, ASA-treated rabbits exhibited significantly greater DHIs and MRI T2 relaxation times than vehicle and untreated control groups. The ASA group had higher mean histologic score than the vehicle group, which demonstrated extensive fiber disorganization and delamination with reduced proteoglycan staining on histology. CONCLUSION: Minimally invasive intervention with intradiscal injection of ASA was successful in reducing IDD in a reproducible rabbit model, with significant improvement in disc height and morphology when compared with vehicle and untreated control groups on radiographic and MRI analyses. LEVEL OF EVIDENCE: N/A.

A percutaneous, minimally invasive annulus fibrosus needle puncture model of intervertebral disc degeneration in rabbits.

PURPOSE: Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. METHODS: Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. RESULTS: There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level ( p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. CONCLUSION: The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. CLINICAL RELEVANCE: The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

Enhanced Skin Regeneration Using a Novel Amniotic-derived Tissue Graft.

BACKGROUND: Chronic and recalcitrant wounds present a significant therapeutic challenge. Amniotic tissues contain many regenerative cytokines, growth factors, and extracellular matrix molecules including proteoglycans, hyaluronic acid, and collagens I, III, and IV. Dehydrated amnion/chorion grafts are currently used to treat a variety of wounds such as diabetic foot ulcers and burns. OBJECTIVE: The investigators hypothesized that processing methodologies, dehydration, and hypothermic processing and storage of amniotic tissues would affect overall quality of wound healing; they compared dehydrated amnion/chorion (dHACM) grafts to a novel hypothermically stored amniotic membrane (HSAM) graft in a full-thickness rat wound model. MATERIALS AND METHODS: Sprague-Dawley rats were anesthetized and prepped for surgery; four 1.5-cm diameter full-thickness wounds were created and treated with either: (1) dHACM, (2) dHACM meshed, (3) HSAM, or (4) wound left ungrafted (sham). After 9 or 21 days, wounds and surrounding areas were collected and stained with hematoxylin and eosin. Blinded quantitative analysis of quality of wound healing was completed by evaluating hair follicle/gland formation, dense/scar-like matrix, and basket-weave matrix. RESULTS: At varying time points following placement of the grafts into full-thickness defects, the authors found that all amniotic-derived tissue grafts appeared to stimulate improved healing over sham wounds, evidenced by more normal-appearing dermal matrix architecture, epidermal structure, and maturity. In addition, the HSAM grafts promoted greater tissue regeneration than the dHACM meshed grafts, as measured by the presence of basket-weave collagen matrix and formation of follicles and glands. CONCLUSIONS: In sum, this study builds on the amassing literature supporting amniotic tissues for wound repair and demonstrates the importance of tissue processing on the quality of wound healing.