Combined nucleus pulposus augmentation and annulus fibrosus repair prevents acute intervertebral disc degeneration after discectomy.

Tissue-engineered approaches for the treatment of early-stage intervertebral disc degeneration have shown promise in preclinical studies. However, none of these therapies has been approved for clinical use, in part because each therapy targets only one aspect of the intervertebral disc's composite structure. At present, there is no reliable method to prevent intervertebral disc degeneration after herniation and subsequent discectomy. Here, we demonstrate the prevention of degeneration and maintenance of mechanical function in the ovine lumbar spine after discectomy by combining strategies for nucleus pulposus augmentation using hyaluronic acid injection and repair of the annulus fibrosus using a photocrosslinked collagen patch. This combined approach healed annulus fibrosus defects, restored nucleus pulposus hydration, and maintained native torsional and compressive stiffness up to 6 weeks after injury. These data demonstrate the necessity of a combined strategy for arresting intervertebral disc degeneration and support further translation of combinatorial interventions to treat herniations in the human spine.

Protective effect of a new hyaluronic acid -carnosine conjugate on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis.

Several studies demonstrated the pharmacological actions of carnosine as well as hyaluronic acid (HA) during joint inflammation. In that regard, the aim of this study was to investigate the protective effect of a new HA -Carnosine conjugate (FidHycarn) on the modulation of the inflammatory response in mice subjected to collagen-induced arthritis (CIA). CIA was induced by two intradermal injections of 100 µl of an emulsion of collagen (CII) and complete Freund's adjuvant (CFA) at the base of the tail on day 0 and 21. At 35 day post CIA induction, the animals were sacrificed. CII injection caused erythema and edema in the hind paws, histological alterations with erosion of the joint cartilage as well as behavioral changes. Oral treatment with FidHycarn starting at the onset of arthritis (day 25) ameliorated the clinical signs, improved behavioral deficits as well as decreased histological and radiographic alterations. The degree of oxidative damage evaluated by inducible nitric oxide synthase (iNOS), nitrotyrosine, poly-ADP-ribose (PAR) expressions and malondialdehyde (MDA) levels, was also significantly reduced in Carnosine+HA association and FidHycarn treated mice. Moreover, the levels of proinflammatory cytokines and chemokines and cyclo-oxygenase COX-2 enzyme were also more significantly reduced by Carnosine+HA and FidHycarn compared to carnosine alone. However, interestingly, in some cases, the effects of FidHycarn were more important than Carnosine+HA association and not statistically different to methotrexate (MTX) used as positive control. Thus, the conjugation of Carnosine with HA (FidHycarn) could represent an interesting therapeutic strategy to combat arthritis disorders.

The relationship between synovial inflammation, structural pathology, and pain in post-traumatic osteoarthritis: differential effect of stem cell and hyaluronan treatment.

BACKGROUND: Synovitis is implicated in the severity and progression of pain and structural pathology of osteoarthritis (OA). Increases in inflammatory or immune cell subpopulations including macrophages and lymphocytes have been reported in OA synovium, but how the particular subpopulations influence symptomatic or structural OA disease progression is unclear. Two therapies, hyaluronan (HA) and mesenchymal stem cells (MSCs), have demonstrated efficacy in some clinical settings: HA acting as device to improve joint function and provide pain relief, while MSCs may have immunomodulatory and disease-modifying effects. We used these agents to investigate whether changes in pain sensitization or structural damage were linked to modulation of the synovial inflammatory response in post-traumatic OA. METHODS: Skeletally mature C57BL6 male mice underwent medial-meniscal destabilisation (DMM) surgery followed by intra-articular injection of saline, a hyaluronan hexadecylamide derivative (Hymovis), bone marrow-derived stem cells (MSCs), or MSC + Hymovis. We quantified the progression of OA-related cartilage, subchondral bone and synovial histopathology, and associated pain sensitization (tactile allodynia). Synovial lymphocytes, monocyte/macrophages and their subpopulations were quantified by fluorescent-activated cell sorting (FACS), and the expression of key inflammatory mediators and catabolic enzyme genes quantified by real-time polymerase chain reaction (PCR). RESULTS: MSC but not Hymovis significantly reduced late-stage (12-week post-DMM) cartilage proteoglycan loss and structural damage. Allodynia was initially reduced by both treatments but significantly better at 8 and 12 weeks by Hymovis. Chondroprotection by MSCs was not associated with specific changes in synovial inflammatory cell populations but rather regulation of post-injury synovial Adamts4, Adamts5, Mmp3, and Mmp9 expression. Reduced acute post-injury allodynia with all treatments coincided with decreased synovial macrophage and T cell numbers, while longer-term effect on pain sensitization with Hymovis was associated with increased M2c macrophages. CONCLUSIONS: This therapeutic study in mice demonstrated a poor correlation between cartilage, bone or synovium (histo)pathology, and pain sensitization. Changes in the specific synovial inflammatory cell subpopulations may be associated with chronic OA pain sensitization, and a novel target for symptomatic treatment.

Efficacy of HYADD®4-G single intra-discal injections in a rabbit model of intervertebral disc degeneration.

BACKGROUND: Various biomaterials/technologies have been tested for treatment of intervertebral disc (IVD) degeneration (IDD). Only few non-surgical options exist. OBJECTIVE: Assessment of efficacy and safety of the hyaluronic acid derivative hydrogel HYADD®4-G in IDD using a well-established rabbit annular puncture model. METHODS: Rabbits were punctured at two IVDs to induce IDD. Thirty days after, IVDs were injected with HYADD®4-G or saline. IVD hydration, height, appearance and tissue organization were assessed by radiographs, MRI and histopathology. Safety of HYADD®4-G injection was evaluated in non-punctured IVDs. RESULTS: HYADD®4-G injection restored disc height to over 75% of the pre-punctured disc, saline injections led to 50% of initial disc height. Compared to saline, HYADD®4-G treatment resulted in improved water retention as revealed by MRI quantification. 83.3% of HYADD®4-G injected discs had normal appearance and reached grade I of the Pfirrmann scale. Regarding tissue organization and cellularity, HYADD®4-G treatment resulted in significantly lower IDD scores than saline (p < 0.01). HYADD®4-G injected into healthy IVDs did not induce inflammation or foreign body reactions. CONCLUSIONS: Intra-discal HYADD®4-G injection is safe and has therapeutic benefits: IDD could be limited through restoration of disc height and hydration and maintenance of normal IVD tissue organization.

Specific concentration of hyaluronan amide derivative induces osteogenic mineralization of human mesenchymal stromal cells: Evidence of RUNX2 and COL1A1 genes modulation.

Hyaluronic acid (HA) is an ideal material for tissue regeneration. The aim of this study was to investigate whether a hyaluronan amide derivative (HAD) can enhance the mineralization of human mesenchymal stem cells (hMSCs). Osteogenically induced hMSCs cultured with or without HAD at different concentrations (0.5 mg/ml or 1 mg/ml) were analyzed for mineral matrix deposition, metabolic activity, cellular proliferation, and the expression of 14 osteogenic genes. Unmodified HA (HYAL) was used as control. We demonstrated that only cells treated daily until day 28 with 0.5 mg/ml HAD, but not with 1 mg/ml of HAD and HYAL, showed a significant induction of mineralization at day 14 compared to the osteogenic control group. HAD at both concentrations tested, significantly decreased the expression of the proliferating marker MKI67 at day 2. By contrast, increased metabolic activity was induced only by HYAL from day 14. HAD at both concentrations significantly down modulated SNAI2, DLX5, RUNX2, COL1A1, and IBSP genes, while significantly up regulated COL15A1. The induction of mineralization of 0.5 mg/ml of HAD at day 14 was significantly dependent on a specific modulation of RUNX2 and COL1A1. Our data demonstrate that only 0.5 mg/ml of HAD, but not HYAL, modulated hMSCs osteogenic differentiation, suggesting that the physicochemical features and concentration of HA products could differently affect osteogenic maturation.

Short-Term Homing of Hyaluronan-Primed Cells: Therapeutic Implications for Osteoarthritis Treatment.

The evaluation of key factors modulating cell homing following injection can provide new insights in the comprehension of unsolved biological questions about the use of cell therapies for osteoarthritis (OA). The main purpose of this in vivo study was to investigate the biodistribution of an intra-articular injection of mesenchymal stromal cells (MSCs) and bone marrow concentrate (BMC) in a rabbit OA model and whether the additional use of sodium hyaluronate (HA) could modulate their migration and delay joint degeneration. OA was surgically induced in adult male New Zealand rabbits. A group of animals was used to test the biodistribution of labeled cells alone or with HA at 7 and 14 days to investigate cell migration. The efficacy of treatments was evaluated in other experimental groups at 2 months. Histology and immunohistochemistry for markers identifying anabolic and catabolic processes in the cartilage and meniscus, or macrophage subset population in the synovial membrane, were performed. Kruskal-Wallis test, followed by post hoc Dunn's test, and Spearman's rank-order correlation method were used. MSCs and BMC preferentially migrate toward tissue areas showing OA features in the meniscus and cartilage and in detail near inflammatory zones in the synovial membrane. The combination with HA contributed to boost cell migration toward articular cartilage. In general, both labeled cells combined with HA were found near cell cluster and fissures in the cartilage and meniscus, respectively, and close to areas of synovial membrane showing mainly anti-inflammatory macrophages. A promotion of joint repair was observed at different levels for all treatments, although BMC-HA treatment resulted as the best strategy to support joint repair. This last, displayed a good protein expression of type II collagen in the cartilage, as well as the presence of anti-inflammatory macrophages in the synovial membrane at 2 months from the treatment. Studies tracking cell biodistribution indicate that priming progenitor cells with HA modulated cell homing favoring not only attachment but also their integration within articular cartilage.

Therapeutic efficacy of intra-articular hyaluronan derivative and platelet-rich plasma in mice following axial tibial loading.

OBJECTIVE: To investigate the therapeutic potential of intra-articular hyaluronan-derivative HYADD® 4-G and/or platelet-rich plasma (PRP) in a mouse model of non-invasive joint injury. METHODS: Non-invasive axial tibial loading was used to induce joint injury in 10-week-old C57BL/6J mice (n = 86). Mice underwent a single loading of either 6 Newton (N) or 9N axial tibial compression. HYADD® 4-G was injected intra-articularly at 8 mg/mL or 15 mg/mL either before or after loading with or without PRP. Phosphate-buffered-saline was injected as control. Knee joints were harvested at 5 or 56 days post-loading and prepared for micro-computed tomography scanning and subsequently processed for histology. Immunostaining was performed for aggrecan to monitor its distribution, for CD44 to monitor chondrocyte reactive changes and for COMP (cartilage oligomeric matrix protein) as an index for cartilage matrix changes related to loading and cartilage injury. TUNEL assay was performed to identify chondrocyte apoptosis. RESULTS: Loading initiated cartilage proteoglycan loss and chondrocyte apoptosis within 5 days with slowly progressive post-traumatic osteoarthritis (no cartilage degeneration, but increased synovitis and ectopic calcification after 9N loading) at 56 days. Mice treated with repeated HYADD® 4-G (15 mg/mL) or HYADD® 4-G (8 mg/mL) ± PRP or PRP alone exhibited no significant improvement in the short-term (5 days) and long-term (56 days) consequences of joint loading except for a trend for improved bone changes compared to non-loaded joints. CONCLUSION: While we failed to show an overall effect of intra-articular delivery of hyaluronan-derivative and/or PRP in reversing/protecting the pathological events in cartilage and synovium following joint injury, some bone alterations were relatively less severe with hyaluronan-derivative at higher concentration or in association with PRP.

Age-independent effects of hyaluronan amide derivative and growth hormone on human osteoarthritic chondrocytes.

AIM: Increased age is the most prominent risk factor for the initiation and progression of osteoarthritis (OA). The effects of human growth hormone (hGH) combined or not with hyaluronan amide derivative (HAD) were evaluated on human OA chondrocytes, to define their biological action and potentiality in OA treatment. MATERIAL AND METHODS: Cell viability, metabolic activity, gene expression and factors released were tested at different time points on chondrocytes treated with different concentrations of hGH (0.01-10 µg/ml) alone or in combination with HAD (1 mg/ml). RESULTS: We found that OA chondrocytes express GH receptor and that the different doses of hGH tested did not affect cell viability, metabolic activity or the expression of collagen type 2, 1, or 10 nor did it induce the release of IGF-1 or FGF-2. Conversely, hGH treatment increased the expression of hyaluronan receptor CD44. HAD combined with hGH reduced metabolic activity, IL6 release and gene expression, but not the suppressor of cytokine signaling 2 (SOCS2), which was significantly induced and translocated into the nucleus. The parameters analyzed, independently of the treatments used proportionally decreased with increasing age of the patients. CONCLUSIONS: hGH only induced CD44 receptor on OA chondrocytes but did not affect other parameters, such as chondrocytic gene markers or IGF-1 or FGF-2 release. HAD reduced all the effects induced by hGH partially through a significant induction of SOCS2. These data show that GH or HAD treatment does not influence the response of the OA chondrocytes, thus the modulation of cellular response is age-independent.

A hexadecylamide derivative of hyaluronan (HYMOVIS®) has superior beneficial effects on human osteoarthritic chondrocytes and synoviocytes than unmodified hyaluronan.

BACKGROUND: Intra-articular hyaluronan (HA) injection provides symptomatic benefit in the treatment of osteoarthritis (OA). Previously we found superior beneficial effects in a large animal OA model of a hexadecylamide derivative compared with unmodified HA of the same initial molecular weight. The current study sought to define possible molecular mechanisms whereby this enhanced relief of symptoms was occurring. METHODS: Chondrocytes and synovial fibroblasts were isolated from tissues of patients undergoing arthroplasty for knee OA. Monolayer cultures of cells were treated with 0, 0.5, 1.0 or 1.5 mg/mL of unmodified HA (500-730 kDa) or a hexadecylamide derivative of HA of the same initial molecular weight (HYADD4®-G; HYMOVIS®) simultaneously or 1 hour before incubation with interleukin (IL)-1beta (2 ng/mL). Cultures were terminated 15 or 30 minutes later (chondrocytes and synovial fibroblasts, respectively) for quantitation of phosphorylated-(p)-JNK, p-NFkappaB, p-p38, or at 24 hours for quantitation of gene expression (MMP1 &13, ADAMTS4 &5, TIMP1 &3, CD44, COL1A1 &2A1, ACAN, PTGS2, IL6, TNF) and matrix metalloproteinase (MMP)-13 activity. RESULTS: The hexadecylamide derivative of HA had significantly better amelioration of IL-1beta-induced gene expression of key matrix degrading enzymes (MMP1, MMP13, ADAMTS5), and inflammatory mediators (IL6, PTGS2) by human OA chondrocytes and synovial fibroblasts. Pre-incubation of cells with the derivatized HA for 1 hour prior to IL-1beta exposure significantly augmented the inhibition of MMP1, MMP13, ADAMTS4 and IL6 expression by chondrocytes. The reduction in MMP13 mRNA by the amide derivative of HA was mirrored in reduced MMP-13 protein and enzyme activity in IL-1beta-stimulated chondrocytes. This was associated in part with a greater inhibition of phosphorylation of the cell signalling molecules JNK, p38 and NF-kappaB. CONCLUSIONS: The present studies have demonstrated several potential key mechanisms whereby the intra-articular injection of a hexadecylamide derivative of HA may be acting in joints with OA.

Efficacy Evaluation of a New Hyaluronan Derivative HYADD® 4-G to Maintain Cartilage Integrity in a Rabbit Model of Osteoarthritis.

Objective: To test the efficacy of a hyaluronan derivative (HYADD®4-G) in a model of osteoarthritis (anterior cruciate ligament [ACLT]) and to compare its efficacy with the injection of growth factors. Design: In a first experimental set-up, specially selected for treatment scheme with published studies on hyaluronan or growth factor efficacy in osteoarthritis, saline, HYADD®4-G, rh-BMP-7, and the treatments of rh-BMP-7 or rh-BMP-2 with HYADD®4-G were injected after ACLT, for five times starting 3 weeks after ACLT. Euthanasia was at day 70. The knees were evaluated by gross morphological observation, x-ray, and histology (Study A). In a second experimental set-up selected to evaluate the efficacy of three viscosupplement injections, starting 4 weeks after ACTL, HYADD®4-G was compared to saline (Study B). Results: (A) X-ray analysis showed more damage in the saline group than all other treatment groups (2.67 ± 0.61 for saline, 0.83 ± 0.26 for HYADD®4-G, 1.67 ± 0.82 for HYADD®4-G with rh-BMP-2, 0.75 ± 0.76 for HYADD®4-G with rh-BMP-7, and 1.58 ± 0.49 for rh-BMP-7), P < 0.05. In the femoral condyle, the Mankin's score for HYADD®4-G with rh-BMP-2, HYADD®4-G with rh-BMP-7, and rh-BMP7 alone was statistically lower compared to saline in the medial part; in the lateral part a significant lower value was observed in the HYADD®4-G with the rh-BMP-2 group. (B) The Kellgren and Lawrence score and Mankin's score was lower in the HYADD®4-G group than in the saline group (P < 0.002 and P = 0.0031). Conclusions: These two studies suggest that HYADD®4-G delayed the cartilage degeneration and that the association of HYADD®4-G with growth factors is synergistic.

In vitro response of osteoarthritic chondrocytes and fibroblast-like synoviocytes to a 500-730 kDa hyaluronan amide derivative.

The aim of this study was to compare the effects of native hyaluronan (HA) with that of its hexadecylamide derivative (HYADD) on proliferation of fibroblast-like synoviocytes (FLS) and chondrocytes. The production of inflammatory and anti-inflammatory cytokines was also analyzed in FLS cultures. The proliferation of osteoarthritis (OA) chondrocytes was enhanced when cells were treated with 0.5-1.5 mg mL(-1) of HA or HYADD®4-G. This effect was completely suppressed by the anti-CD44 antibody. At 0.5 to 1 mg mL(-1) , HA and HYADD®4-G did not influence the proliferation of normal or pathological FLS; however, at the higher concentration (1.5 mg mL(-1) ), HYADD®4-G did significantly inhibit cell proliferation. As to effects on inflammation, a significant increase in the expression of the IL-10 gene was observed when FLS were pretreated with tumor necrosis factor alpha and then cultured in the presence of 0.5 mg mL(-1) HYADD® 4-G or HA. The effects of HA derivatives on FLS proliferation and production of anti-inflammatory cytokines indicate that they may be of therapeutic benefit in OA. The longer residence time in the joint cavity, the increased viscoelasticity, and the anti-inflammatory potential of HYADD®4-G make it a better candidate than native HA for OA therapy.

Effective lubrication of articular cartilage by an amphiphilic hyaluronic acid derivative.

BACKGROUND: Intra-articular injection of hyaluronic acid based therapies is gaining popularity as a treatment option for non-operative management of patients with symptomatic osteoarthritis. Although there is an abundance of evidence for both biological and mechanical mechanisms of joint protection by hyaluronic acid, one clear intention of viscosupplementation is to reduce friction and wear by providing an extrinsic lubricant. We tested the in vitro friction response of a novel hyaluronic acid derivative that presents amphiphilic features to promote adhesion to the cartilage surface and thereby improve cartilage lubrication. METHODS: Migrating Contact Area and Static Contact Area friction tests were conducted on bovine articular cartilage to assess the efficacy of two lubricants, a chemically modified amphiphilic hyaluronic acid and synovial fluid from a healthy joint, as well as a phosphate buffered saline negative control. FINDINGS: No differences in lubrication (P=0.34) were evident between the three test articles during the Migrating Contact Area test, which represents articulation of healthy articular cartilage. The modified hyaluronic acid presented an equilibrium friction coefficient 2.8 times less than that of the synovial fluid (P ≤ 0.0005) and five times less than that of the PBS control (P ≤ 0.0001) during the Static Contact Area test, representing a mixed lubrication condition. INTERPRETATION: The present study demonstrated that a chemically modified amphiphilic hyaluronic acid can provide equivalent lubrication to synovial fluid during articulation of loaded healthy articular cartilage and can provide superior lubrication as indicated by a lower coefficient of friction than synovial fluid under loading conditions potentially associated with cartilage wear.