Objectives. Intra-articular injections of local anaesthetics (LA), glucocorticoids (GC), or hyaluronic acid (HA) are used to treat osteoarthritis (OA). Contrast agents (CA) are needed to prove successful intra-articular injection or aspiration, or to visualize articular structures dynamically during fluoroscopy. Tranexamic acid (TA) is used to control haemostasis and prevent excessive intra-articular bleeding. Despite their common usage, little is known about the cytotoxicity of common drugs injected into joints. Thus, the aim of our study was to investigate the effects of LA, GC, HA, CA, and TA on the viability of primary human chondrocytes and tenocytes in vitro. Methods. Human chondrocytes and tenocytes were cultured in a medium with three different drug dilutions (1:2; 1:10; 1:100). The following drugs were used to investigate cytotoxicity: lidocaine hydrochloride 1%; bupivacaine 0.5%; triamcinolone acetonide; dexamethasone 21-palmitate; TA; iodine contrast media; HA; and distilled water. Normal saline served as a control. After an incubation period of 24 hours, cell numbers and morphology were assessed. Results. Using LA or GC, especially triamcinolone acetonide, a dilution of 1:100 resulted in only a moderate reduction of viability, while a dilution of 1:10 showed significantly fewer cell counts. TA and CA reduced viability significantly at a dilution of 1:2. Higher dilutions did not affect viability. Notably, HA showed no effects of cytotoxicity in all drug dilutions. Conclusion. The toxicity of common intra-articular injectable drugs, assessed by cell viability, is mainly dependent on the dilution of the drug being tested. LA are particularly toxic, whereas HA did not affect cell viability. Cite this article: P. Busse, C. Vater, M. Stiehler, J. Nowotny, P. Kasten, H. Bretschneider, S. B. Goodman, M. Gelinsky, S. Zwingenberger. Cytotoxicity of drugs injected into joints in orthopaedics. Bone Joint Res 2019;8:41–48. DOI: 10.1302/2046-3758.82.BJR-2018-0099.R1

The extracellular matrix (ECM)-based biomaterials provide a platform to mimic the disc microenvironment in facilitating stem cell transplantation for tissue regeneration. However, little is known about in vitro preconditioning human umbilical cord Wharton Jelly-derived mesenchymal stem cells (MSCs) on 3D hyaluronic acid (HA)/type II collagen (COLII) hydrogel for nucleus pulposus (NP) phenotype and pain modulation. We developed a tuneable 3D HA/COLII by fabricating HA/COLII hydrogel at 2 mg/ml COLII and various weight ratios of HA:COLII, 1:9 and 4.5:9. The hydrogel was characterized for degradability, stability, and swelling capacity. The viability of hWJ-MSC encapsulated on hydrogel supplemented with TGF-β3 was assessed. The implantation of HA/COLII hydrogel was done in surgically induced disc injury model of pain in the rat tail. The general health status in rats was monitored. The nociceptive behaviour in rats was performed for mechanical allodynia using von Frey test. The HA/COLII 4.5:9 hydrogel showed higher swelling capacity than weight ratio 1:9, suggesting that a higher amount of HA can absorb a large amount of water. Both HA/COLII 4.5:9 and 1:9 hydrogel formulations had a similar degradation profile, stable to the hydrolytic process. The hWJ-MSC-encapsulated on hydrogel marked higher cell viability with round morphology shape of cells in vitro. The surgically induced disc injury in the rat tail evoked mechanical allodynia, without affecting general health status in rats. The implantation of HA/COLII 1:9 hydrogel was observed to slightly alleviate injury-induced mechanical allodynia. Fine-tuning HA/COLII-based hydrogel provides the optimal swelling capacity, stability, degradability, and non-cytotoxic, mimicking the 3D NP niche in guiding hWJ-MSCs towards NP phenotype. The HA/COLII hydrogel could be employed as an advanced cell delivery system in facilitating stem cell transplantation for intervertebral disc regeneration targeting pain

Objective. To study the effect of hyaluronic acid (HA) on local anaesthetic chondrotoxicity in vitro. Methods. Chondrocytes were harvested from bovine femoral condyle cartilage and isolated using collagenase-containing media. At 24 hours after seeding 15 000 cells per well onto a 96-well plate, chondrocytes were treated with media (DMEM/F12 + ITS), PBS, 1:1 lidocaine (2%):PBS, 1:1 bupivacaine (0.5%):PBS, 1:1 lidocaine (2%):HA, 1:1 bupivacaine (0. 5%):HA, or 1:1 HA:PBS for one hour. Following treatment, groups had conditions removed and 24-hour incubation. Cell viability was assessed using PrestoBlue and confirmed visually using fluorescence microscopy. Results. Media-treated groups had a mean of 1.55×10. 4. cells/well (. sem. 783). All treated cells showed statistically significant reduced viability when compared with media alone (all p < 0.003). Cells treated with bupivacaine + HA (6.70×10. 3. cells/well (. sem. 1.10×10. 3. )) survived significantly more than bupivacaine (2.44×10. 3. cells/well (. sem . 830)) (p < 0.001). Lidocaine + HA (1.45×10. 3. cells/well (. sem. 596)) was not significantly more cytotoxic than lidocaine (2.24×10. 3. cells/well (. sem. 341)) (p = 0.999). There was no statistical difference between the chondrotoxicities of PBS (8.49×10. 3. cells/well (. sem. 730) cells/well) and HA (4.75×10. 3. cells/well (. sem. 886)) (p = 0.294). Conclusions. HA co-administration reduced anaesthetic cytotoxicity with bupivacaine but not lidocaine, suggesting different mechanisms of injury between the two. Co-administered intra-articular injections of HA with bupivacaine, but not lidocaine, may protect articular chondrocytes from local anaesthetic-associated death. Cite this article: Bone Joint Res 2013;2:270–5

In this study, we developed biocompatible adhesive which enables implanted chondrogenic-enhanced hASCs being strongly fixed to the lesion site of defected cartilage. The bioengineered mussel adhesive protein (MAP) was produced and purified using a bacterial expression system as previously reported. The cell encapsulated coacervate was formulated with two polyelectrolyte, the MAP and 723kDa hyaluronic acid (HA). MAP formed liquid microdroplets with HA and subsequently gelated into microparticles, which is highly viscous and strongly adhesive. The MAP with chondro-induced hASCs were implanted on the osteochondral defect created in the patellar groove/condyle of OA-induced rabbits. Rabbits were allocated to three different groups as follows: Group1 – Fibrin only; Group2 – Fibrin with hASCs (1.5×10. 6. chondro-induced hASCs); Group3; MAP with hASCs. The implanted cells were labeled with a fluorescent dye for in vivo visualization. After 35 days, fluorescent signals were more potently detected for MAP with hASCs group than Fibrin with hASCs group in osteochondral defect model. Moreover, histological assessment showed that MAP with hASCs group had the best healing and covered with hyaline cartilage-like tissue. The staining image shows that MAP with hASCs group were filled with perfectly differentiated chondrocytes. Although Fibrin with hASCs group had better healing than fibrin only group, it was filled with fibrous cartilage which owes its flexibility and toughness. As MAP with hASCs group has higher possibility of differentiating to complete cartilage, Fibrin only group and Fibrin with hASCs group have failed to treat OA by rehabilitating cartilage. In order to clarify the evidence of remaining human cell proving efficacy of newly developed bioadhesive, human nuclear staining was proceeded with sectioned rabbit cartilage tissue. The results explicitly showed MAP with hASCs group have retained more human cells than Fibrin only and Fibrin with hASCs groups. We investigated the waterproof bioadhesive supporting transplanted cells to attach to defect lengthily in harsh environment, which prevents cells from leaked to other region of cartilage. Collectively, the newly developed bio-adhesive, MAP, could be successfully applied in OA treatment as a waterproof bioadhesive with the capability of the strong adhesion to target defect sites

Hyaluronic acid (HA) is responsible for the viscoelastic properties of synovial fluid and cartilage. Compared to healthy joints, synovial fluid in osteoarthritic joints contains HA of lower concentration and molecular weight. Hyaluronic acid hybrid complexes are composed by long and short HA chains linked by H bonds. These rheological characteristics and viscoelastic properties were produced by thermal patented process without chemical modification. Chondroitin sulfate (CS) is one of the essential components of the articular cartilage matrix and plays a key role in cartilage's mechanical and elastic properties. Biotechnological chondroitin (CB) is produced through fermentative/biotechnological processes and, unlike CS, is not sulfated. It has been shown that CB to play a more significant role in the phenotypic maintenance of chondrocytes than chondroitin sulfate and increases their viability and proliferation. A recent A Single-Arm, Open-Label, Pilot Study was conducted to evaluate the safety and efficacy of a single-dose intra-articular injection of Hybrid Hyaluronic acid and Sodium Chondroitin in the Treatment of Symptomatic Hip Osteoarthritis. A single injection of HS-SC was well tolerated and safe in the treatment of symptomatic hip OA. The treatment demonstrated a rapid significant improvement in pain (VAS) and function (Lequesne's Index) up to 6 months of follow-up

Abstract. Objectives. Bone marrow aspirate concentrate (BMAC), together with fibrin glue (Tisseel, Baxter, UK) and Hyaluronic acid (HA) were used as a one-step cell therapy treating patients with ankle cartilage defects in our hospital. This therapy was proven to be safe, with patients demonstrating a significant improvement 12 months post-treatment. Enriched mesenchymal stem cells (MSCs) in BMAC are suggested inducers of cartilage regeneration, however, currently there is no point-of-care assessment for BMAC quality; especially regarding the proportion of MSCs within. This study aims to characterise the cellular component of CCR-generated BMAC using a point-of-care device, and to investigate if the total nucleated cell (TNC) count and patient age are predictive of MSC concentration. Methods. During surgery, 35ml of bone marrow aspirate (BMA) was collected from each patients’ iliac crest under anaesthesia, and BMAC was obtained via a commercial kit (Cartilage Regeneration kit, CCR, Innotec. ®. , UK). BMAC was then mixed with thrombin (B+T) for injection with HA and fibrinogen. In our study, donor-matched BMA, BMAC and B+T were obtained from consented patients (n=12, age 41 ± 16years) undergoing surgery with BMAC therapy. TNC, red blood cell (RBC) and platelet (PLT) counts were measured via a haematology analyser (ABX Micros ES 60, Horiba, UK), and the proportion of MSCs in BMA, BMAC and B+T were assessed via colony forming unit-fibroblast (CFU-F) assays. Significant differences data in matched donors were tested using Friedman test. All data were shown as mean ± SD. Results. Mean TNC counts in BMA and BMAC were not significantly different (14.0 ± 4.4 million/ml and 19.4 ± 32.9 million/ml, respectively, P>0.9999). However, TNC counts were significantly lower in B+T compared to BMAC (9.7 ± 24.5 million/ml and 19.4 ± 32.9 million/ml, respectively, P=0.0167). Similarly, PLT counts were decreased in B+T compared to BMAC (40.7 ± 30.7 million/ml and 417.5 ± 365.5 million/ml, respectively, P<0.0001), however, PLTs were significantly concentrated in BMAC compared to BMA (417.5 ± 365.5 million/ml and 114.8 ± 61.6 million/ml, respectively, P=0.0429). RBC counts were significantly decreased in BMAC and B+T compared to BMA (P=0.0322 and P<0.0001, respectively). Higher concentration of MSCs were observed in BMAC compared to BMA (0.006% ± 0.01% and 0.00007% ± 0.0001%, respectively, P=0.0176). Similar to TNCs and PLTs, the proportion of MSCs significantly decreased in B+T compared to BMAC (0.0004% ± 0.001% and 0.006% ± 0.01%, respectively, P=0.0023). Furthermore, patient age and TNC counts did not correlate with MSC concentration (Spearman's Rank test, P=0.3266 and P=0.4880, respectively). Conclusions. BMAC successfully concentrated PLTs, but BMAC preparations were highly variable. Mixing BMAC and thrombin however, as described in the CCR protocol, resulted in a dramatic reduction in TNCs, PLTs and MSCs. TNC counts and patient age could not be used to predict the MSC proportion in the BMAC based on current data. Future work aims to look at the biomolecule profile of BMAC plasma, and to correlate them to patient clinical outcomes. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

Introduction and Objective. Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart. ®. product, a combination of HA and SS. Materials and Methods. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart. ®. at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by flow cytometry with MitoSox and dichlorodihydrofluorescein diacetate (DCFDA) assays. Gene expression of inflammation/oxidative stress-related transcripts (MMP1/MMP3/INOS/COX2) was evaluated by qRT-PCR using TBP as reference. Results. NO was detected only in inflamed chondrocytes and Diart® was able to abolish its levels. NO was not detected in synoviocytes in all conditions. IL-1B reduced both cytoplasmic (−66%) and mitochondrial (−68%) ROS in chondrocytes, with Diart® partially restoring (+40%) mitochondrial levels. In synoviocytes, IL-1B did not alter ROS, with Diart® modestly increasing (+27%) mitochondrial levels. Inflammation was able to increase transcript levels of all tested markers, with the exception of INOS in synoviocytes. In chondrocytes, Diart® significantly (p < 0.05) reduced COX2 (−75%) and MMP1 (−33%). In synoviocytes, Diart® significantly reduced COX2 (−77%) and MMP3 (−84%), with MMP1 53% decreased albeit without reaching statistical significance. Conclusions. Diart. ®. biochemical and physiologic properties in the tested in vitro model of inflammation on donor-matched chondrocytes and synoviocytes allowed reducing inflammation and oxidative stress-related markers, prompting the use of this combination as successful strategy to manage OA-related symptoms

Abstract. Objectives. The ability to predict which patients will improve following routine surgeries aimed at preventing the progression of osteoarthritis is needed to aid patients being stratified to receive the most appropriate treatment. This study aimed to investigate the potential of a panel of biomarkers for predicting (prior to treatment) the clinical outcome following treatment with microfracture or osteotomy. Methods. Proteins known to relate to OA severity, with predictive value in autologous cell implantation treatment or that had been identified in proteomic analyses (aggrecanase-1/ ADAMTS-4, cartilage oligomeric matrix protein (COMP), hyaluronic acid (HA), Lymphatic Vessel Endothelial Hyaluronan Receptor-1, matrix metalloproteinases-1 and −3, soluble CD14, S100 calcium binding protein A13 and 14-3-3 protein theta) were assessed in the synovial fluid (SF) of 19 and 13 patients prior to microfracture or osteotomy, respectively, using commercial immunoassays. Levels of COMP and HA were measured in the plasma of these patients. To find predictors of postoperative function, multiple linear regression analyses were performed. Results. Linear regression analyses demonstrated that a lower concentration of HA in pre-operative SF was predictive of improved knee function (higher Lysholm score) following microfracture surgery. Further, lower pre-operative activity of ADAMTS-4 in SF was a significant, independent predictor of higher post-operative Lysholm score (improved joint function) following osteotomy surgery. Conclusion. This study is novel in identifying biomarkers with the potential to predict clinical outcome in patients treated with microfracture or osteotomy of the knee. Lower concentrations of HA and undetectable activity of ADAMTS-4 in the joint fluid of individuals with cartilage defects/early-OA may be used in algorithms to stratify patients to the most appropriate surgery. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The use of stem cells transplanted into the intervertebral disc (IVD) is a promising regenerative approach to treat intervertebral disc degeneration (IDD). The aim of this study was to assess the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) loaded with human mesenchymal stem cells (hMSCs), on IVD extracellular matrix synthesis and nucleus pulposus (NP) marker expression in a whole IVD culture model. HA was blended with batroxobin (BTX), a gelling agent activated in presence of PRP to construct a hydrogel. Bovine IVDs (n=25) were nucleotomised and filled with 1×10. 6. or 2×10. 6. hMSCs suspended in ∼150 mL of the PRP/HA/BTX hydrogel. IVDs harvested at day 0 and nucleotomised IVDs with no hMSCs and/or hydrogel were used as controls. hMSCs alone or encapsulated in the hydrogel were also cultured in well plates to examine the effect of the IVD microenvironment on hMSCs. After 1 week, tissue structure, scaffold integration and gene expression of anabolic (collagen type I, collagen type II and aggrecan), catabolic (matrix metalloproteinase 3 – MMP-3 –, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs 4) and NP cell (cytokeratin 19, carbonic anhydrase 12, cluster of differentiation 24) markers were assessed. Histological analysis showed a good integration of the scaffold within the NP area with cell repopulation. At the gene expression level, the hMSC-loaded hydrogels demonstrated to increase disc cell anabolic and catabolic marker expression and promoted hMSC differentiation towards a NP cell phenotype. This study demonstrated that the HA/PRP/BTX may represent a valid carrier for hMSCs being capable of stimulating cell activity and NP marker expression as well as achieving a good integration with the surrounding tissues

Osteoarthritis (OA) is a joint degenerative disease leading to chronic pain and disability, thus resulting in a major socioeconomic health burden. OA, which has long been believed to be a cartilage disease, is now considered a whole-joint disorder affecting various anatomical structures, including subchondral bone. Hyaluronic Acid (HA) is commonly used as intra-articular viscosupplementation therapy for its mechanical features and biological effects. Bisphosphonates (BPs) are antiresorptive agents inhibiting recruitment and maturation of osteoclast precursors and activity of mature osteoclasts in the bone. Pre-clinical evidences in the literature, show that intra-articular BPs could impact on OA progression, slowing down or reversing it. The combination of HA biological and mechanical role and Alendronate (ALD) antiresorptive effect could be an interesting strategy for OA treatment. This study describes the synthesis and characterization of FID-134, a new chemical derivative of HA conjugated with ALD by means of a covalent bond, cleavable in physiological condition. FID-134 was synthesized starting from 500 kDa HA: chemical structure and functionalization degree with ALD were investigated by NMR and ICP-OES. Kinetics of ALD release from FID-134 was determined in TRIS buffer at 37°C and compared to a simple mixture of HA+ALD. 20mg/mL formulations of FID-134 and HA+ALD were investigated for viscoelastic properties, in absence and presence of Ca. 2+. ions. The cytotoxicity of FID-134 and free ALD were tested on Saos-2 osteoblasts (ATCC HTB-85) and on primary bovine chondrocytes (PBC) at day 1, 3 and 7. The efficacy of FID-134 was assessed in an inflammatory arthritis in vitro model, where bovine cartilage biopsies were exposed to IL-1β/OSM (10ng/mL) for 3 weeks; at the same time, cartilage explants were treated with FID-134. Collagen release in the surnatants was quantified and compared to controls. FID-134 structure was confirmed by NMR and the 20% mol/mol functionalization degree was determined by ICP-OES. Only about 50% of total bound ALD was released from FID-134 within 7 days, resulting slower compared to HA+ALD mixture. In presence of Ca. 2+. ions, viscoelastic properties of FID-134 dramatically improved, while HA+ALD formulation remained unaffected. The cytotoxicity of ALD was evident at 100 μM on Saos-2 and PBC after 3 days, while no cytotoxicity was observed at 7 days with FID-134. In the cartilage explant model, a strong collagen release was detected in inflammatory conditions after 3 weeks; this tendency was reversed, and collagen release halved when FID-134 was added to the biopsies. The synthesized HA-ALD adduct, FID-134, opens the door for a new approach for OA treatment. The results suggest that FID-134 could be beneficial in cartilage degradation and in restoration of subchondral bone function. Finally, local administration and controlled BP release would likely overcome the drawbacks of ALD oral administration, such as unspecific features and long-term toxic side effects

The rheological properties of synovial fluid (SF) are largely attributed to the presence of high molecular weight hyaluronic acid (HA). In normal SF, HA has been shown to be an anti-inflammatory molecule able to increase the viscosity and promote endogenous production of HA. The aim of the present report was to investigate the possible effect of HA concentration in rheological properties (elastic modulus, G´ and viscous modulus, G´´) of osteoarthritic equine SF. For this purpose, SF from intercarpal, metacarpophalangeal and distal interphalangeal joint was aspirated by aseptic arthrocentesis from 60 Warmblood horses. For determining HA concentrations in equine SF samples, a commercially available ELISA kit was used. Additionally, full rheological sample characterization was carried out with an AR-G2 rheometer (TA Instruments Ltd., UK) in order to measure the elastic G´ and viscous G´´ moduli, at horse's body (37.5 ºC) temperature. The ANOVA findings revealed statistically significant main effects of the factors Joint Type (p = 0.001), and main effects of covariates Age (p = 0.019) and HA (p < 0.001) on the mean values of logG” and logG' measurements. Interpreting the coefficients of the covariate HA, a positive correlation of HA was detected on the response logG” and logG' measurements. Collectively, these data illustrate the role of HA in equine pathological SF

Osteoarthritis (OA) is an inflammatory degenerative disease that affects every fourth person with irreversible damage to the articular. Mesenchymal stem cells (MSCs) have been shown to affect host cells by paracrine stimulation in regenerative environments. Here we apply hyaluronic acid (HA), an essential part of the extracellular matrix in cartilage, for MSC immobilization. The aim was to investigate long-term MSC survival and paracrine effect on chondrocytes in an inflammatory co-culture environment. We hypothesized that MSCs immobilized in a HA hydrogel could provide a long-term immunomodulatory effect on chondrocytes in vitro. Human MSCs were seeded in a HA hydrogel and co-cultured with non-osteoarthritic human chondrocytes in biphasic wells inhibiting cellular contact. An inflammatory environment was induced by IL1-beta and compared with standard culture medium. Relative gene expressions of collagen types I, II and X, aggrecan, SOX9, MMP-13 and ADAMTS-5) were examined at day 3,7,14 and 28. Significant up-regulation of SOX9 at day 7, 14 and 28 and a significant down-regulation of ADAMTS-5 (day 14 and 28) was observed with co-culture of HA-immobilized MSCs and MSCs compared with controls with or without HA (without MSCs)No changes in expression was observed for aggrecan and collagen type 1. We showed that MSC affect the expression of SOX9 and ADAMTS-5 in a paracrine manner when co-cultured with chondrocytes in an inflammatory environment. MSCs immobilized in HA hydrogels survived and were contained in the hydrogel for up to 28 days. This suggests that HA-immobilized MSCs could potentially be used as adjuvant treatment of OA

Bone regenerative medicine aims at designing biomimetic biomaterials able to guide stem cells fate towards osteoblast lineage and prevent orthopaedic common pathogen adhesion. Owing to bone inorganic/organic composition, we herein report, using a versatile process based on simultaneous spray coating of interacting species, a calcium phosphate (CaP) / chitosan (CHI) / hyaluronic acid (HA) functionalized collagen membrane as a new strategy for bone regenerative medicine. Physicochemical characterizations of CaP-CHI-HA coating were performed by scanning electron microscopy, X-ray photoelectron and infrared spectroscopies and high-resolution transmission electron microscopy, revealing the formation of a thin coating mainly composed of non-stoichiometric crystalline hydroxyapatite dispersed into polymorphic organic film. Biocompatibility of CaP-CHI-HA coated membrane, evaluated after 7 days in contact with human mesenchymal stem cells (MSCs), showed spread, elongated and aligned cells. Metabolic activity and DNA quantification studies showed an increase in MSCs proliferation on coated membrane compared to uncoated membrane over the study time. Similarly, cytokines (IL-6, IL-8, osteoprotegerin) and growth factors (VEGF, bFGF) release in supernatant, as well as endothelial cells recruitment, were significantly increased in presence of CaP-CHI-HA coated membrane. Thus, CaP-CHI-HA coated membrane provides a suitable environment for MSCs to induce bone healing. Moreover, pro-inflammatory cytokines (IL-1β and TNF-α) secretion by human monocytes was significantly reduced on CaP-CHI-HA coating compared to LPS stimulation. CaP-CHI-HA coating also reduced significantly Staphylococcus aureus and Pseudomonas aeruginosa adhesion on the membrane, conferring a bacterial anti-adhesive surface. Based on our results, CaP-CHI-HA functionalized collagen membrane provides an interesting material for bone regeneration

Tissue engineering is a promising approach to regenerate damaged skeletal tissues. In particular, the use of injectable hydrogels alleviates common issues of poor cell viability and engraftment. However, uncontrolled cell fate, resulting from unphysiological environments and degradation rates, still remain a hurdle and impedes tissue healing. We thus aim at developing a new platform of injectable hyaluronic acid (HA) hydrogels with a large panel of properties (stiffness, degradation…) matching those of skeletal tissues. Hence, HA with different molecular weights were functionalized with silylated moieties. Upon injection, these hydrogels formed through a sol-gel chemistry within 5 to 20 minutes in physiological conditions, as demonstrated by rheological characterization. By varying the crosslinking density and concentration, we obtained hydrogels spanning a large range of elastic moduli (E = 0.1–20 kPa), similar to those of native ECMs, with tunable biodegradation rates (from 24 hours to > 50 days) and swelling ratios (500 to 5000% (w/w)). Cell viability was confirmed by Live/Dead assays and will be completed by in vivo subcutaneous implantations in mice to study the foreign body reaction and degradation rate. We further developed hybrid HA/biphasic calcium phosphate granules hydrogels and demonstrated a strong mechanical reinforcement (E = 0.1 MPa) and a faster relaxation behaviour (τ. 1/2. < 400s), with similar degradation rates. Ongoing in vitro differentiation assays and in vivo implantations in a rabbit femur model will further assess their ability to drive bone regeneration. Collectively, these results suggest that this hydrogel platform offers promising outcomes for improved strategies in skeletal tissue engineering

Objectives. To evaluate the in vitro effects of hyaluronic acid (HA) on adipose-derived stem cells (ASC) in order to consider the possibility of their combined used in the treatment of knee arthrosis. Material and methods. The ASC cells were grown both in the presence and absence of AH, and several studies were carried out: proliferation (WST8) and cell viability studies (Alamar Blue and Trypan Blue), possible chondrogenic differentiation (collagen type 2 expression) by RT-PCR, AH receptor expression (CD44) by flow cytometry and RT-QPCR, and expression of inflammatory and anti-inflammatory factors (IL-6, TGFß, IL-10) by RT-QPCR. Results. The number of ASC significantly increased after 7 days with HA (158±39%, p < .05). Additionally, the cell viability of the ASC treated with HA after 1, 3, 5 and 7 days was similar to that of the control cells, being considered non-toxic. There were no changes observed in the expression of CD44 and chondrogenic differentiation. TGFß expression was not modified after AH treatment, but there was a 4-fold decrease in IL-6 expression and IL-10 expression increased up to 2-fold compared to control cells. Conclusions. Hyaluronic acid favours ASC proliferation without causing cellular toxicity, and inducing an anti-inflammatory profile in these cells. Hyaluronic acid appears to be a suitable vehicle for the intra-articular administration of mesenchymal stem cells

Background. Hyaluronic acid (HA) hydrogels are becoming an increasingly attractive choice for the creation of new biomaterials useful in wound care, tissue engineering and regenerative medicine, because of their high level of biocompatibility and biodegradability, and for their ability to imitate the environment of the extracellular matrix (ECM). Due to the poor biomechanical properties of native hyaluronan, a variety of chemical modifications have been devised to provide mechanically and chemically stiffer materials. Methods. In this work, 200 kDa hyaluronic acid was modified with coumarin moieties via a functional linker (FID119) and photo-polymerised into networks through a [2+2] cycloaddition reaction using near-UV light (l. max. =365 nm). This method allows to obtain “wall-to-wall” hydrogels starting from moderately viscous solutions. FID119 can therefore be deposited in the cartilage defect as an aqueous solution and can be polymerised in situ after UV irradiation. Results. With a HA molar derivatisation ranging from 10% to 40% and a concentration varying from 10 mg/mL to 40 mg/mL, hydrogels exhibited a wide range of physical properties. When a suspension of human dermal fibroblasts was photo-encapsulated within the hydrogels, cells retained a rounded morphology throughout the period of culture and showed no spreading. Cells remained viable after 48 hours encapsulation, confirming that their viability was affected neither by the polymerisation process nor by UV irradiation. In this study we have also evaluated the proliferation of fibroblasts encapsulated in HA-hydrogels at different degree of reticulation, concentrations and polymerisation time. By means of the resazurin reduction assay (Alamar Blue) it has been shown that encapsulated fibroblasts showed overall lower metabolic activity compared to fibroblasts cultured in traditional 2D tissue culture plastic dishes, in all the tested conditions. Conclusions. This work represents a first step towards the development and characterisation of new HA-based advanced biomaterial to be used as scaffolds in cartilage regeneration. The screening of the different FID119 preparations led to the selection of three prototypes representing the best compromise between physical-chemical properties and biocompatibility. Level of Evidence. III

The excursion resistance between the tendon and pulley is an important factor contributing to the limitation of function after surgery to the hand. The administration of hyaluronic acid (HA) in the early rehabilitation after tendon grafting may help to prevent adhesions. We evaluated changes in the excursion resistance between potential sources of flexor tendon grafts and the annular pulley in a canine model after administration of HA. The intrasynovial and extrasynovial tendons were soaked in 10 mg/ml of HA for five minutes. The excursion resistance between these tendons and the annular pulley of an intact proximal phalanx and that of the same tendons of the opposite foot without administration of HA were evaluated. The tendon of flexor digitorum profundus of the second toe without administration of HA was used as a control. The gliding resistance of canine tendons was significantly decreased after the administration of HA especially in the extrasynovial tendons. Our findings suggest that the administration of HA may improve the gliding function of a flexor tendon graft

Summary Statement. Transportation media and injection protocol have implications for the viability of MSCs used for intra-lesional treatment of tendon injuries. Every effort should be made to implant cells within 24h of laboratory re-suspension, using a needle bore larger than 21G. Introduction. Intra-lesional implantation of autologous mesenchymal stem cells (MSCs) has resulted in significant improvements in tendon healing in experimental animal models. Intra-tendinous injection of MSCs into naturally-occurring equine tendon injuries has been shown to be both safe and efficacious. 1. and these protocols can assist in the translation to the human. Efficient transfer of cells from the laboratory into the tissue requires well validated techniques for transportation and implantation. The aim of this study was to determine the influence of transport media and injection procedure on cellular damage. Methods. Bone marrow derived MSCs (n=3 horses) were prepared and expanded as described. 1. Cells were suspended in 0.5mL of experimental media at 2.5×10. 6. cells/mL and stored at 4–8°C for 24, 48 and 72h. Experimental media were: bone marrow aspirate (BMA); cell culture media (DMEM); equine serum; equine plasma; isotonic saline; hyaluronic acid (HA); platelet-rich plasma (PRP) and frozen (in 90% serum, 10% DMSO). In addition, cells suspended in DMEM were injected through a 19G, 21G or 23G needle and cell viability, proliferation and apoptosis were analysed using trypan blue, alamarBlue® and Annexin-V assays respectively. Results. There was no significant difference in overall viability at 24h storage in any media, however cell death was most rapid when cells were suspended in BMA, PRP and serum. Viability was greatest at all time points when cells were frozen. Cell proliferation was similar following storage for 24 and 72h in all media, except for 24h in serum, wherein proliferation was enhanced. There was no significant decrease in viability immediately following injection but 21G and 23G needles resulted in a marked increase in apoptotic cells compared to 19G and non-injected controls after 24h when re-seeded for culture. All needle gauges resulted in a marked decrease in cell proliferation immediately post-injection with recovery by 2h post-injection. Conclusions. Although there is, as yet, no guidance on the storage of MSCs, it has been suggested that in vitro storage of hematopoietic stem or progenitor cells should not exceed 2h. 2. This suggestion is impractical both for current equine therapeutic use and when considering future, commercial applications of MSC therapy in humans, because of the necessity to transport the cells from a remote licensed facility to the clinic. Our data suggest an upper limit of 24h for transportation, whereas for transportation of greater duration than 24h, cells should ideally be frozen, to maximise viability. An increased number of dead cells potentially has two adverse consequences; first, a reduced efficacy and second, the presence of dead cell debris may induce inflammation. While the first can be compensated for by higher cell numbers, this compounds the problems of the second. This study reinforces the importance of limiting the delay between preparation of cells for shipment from the laboratory and implantation in the clinic and suggests that an injection procedure while not causing immediate cell death can cause significant delayed cell death if small bore needles are used

Summary. Coating of titanium implants with BMP-2-loaded polyelectrolyte multilayer films conferred the implant surface with osteoinductive properties which are fully preserved upon both air-dried storage and γ-sterilization. Although BMP-2 is recognised as an important molecule for bone regeneration, its supraphysiological doses currently used in clinical practice has raised serious concerns about cost-effectiveness and safety issues. Thus, there is a strong motivation to engineer new delivery systems or to provide already approved materials with new functionalities. Immobilizing the growth factor onto the surface of implants would reduce protein diffusion and increase residence time at the implantation site. To date, modifying the surfaces of metal materials, such as titanium or titanium alloys, at the nanometer scale for achieving dependable, consistent and long-term osseointegration remains a challenging approach. In this context, we have developed an osteoinductive coating of a porous titanium implant using biomimetic polyelectrolyte multilayer (PEM) films used as carriers of BMP-2. The PEM films were prepared by alternate deposition of 24 layer pairs of poly(L-lysine) (PLL) and hyaluronic acid (HA) layers (∼3.5 µm in thickness); such films were then cross-linked by means of a water-soluble carbodiimide (EDC) at different degrees. The amount of BMP-2 loaded in these films was tuned (ranging from 1.4 to 14.3 µg/cm. 2. ) depending on the cross-linking extent of the film and of the BMP-2 initial concentration. Because packaging, and storage of the devices are important issues that may limit a wide application of biologically functionalised materials, we assessed in the present study the osteoinductive performance of the BMP-2 loaded PEM coatings onto custom-made 3D porous scaffolds made of Ti-6Al-4V in vitro and in vivo pertinent to long-term storage in a dry state and to sterilization by gamma irradiation. Analysis of PEM films by infrared spectroscopy evidenced that the air-dried films were stable for at least one year of storage at 4°C and they resisted exposure to γ-irradiation at clinically approved doses. The preservation of the growth factor bioactivity was evaluated both in vitro (using C2C12 cell model) and in vivo (in a rat ectopic model). In vitro, BMP-2 loaded in dried PEM films exhibited shelf-life stability at 4°C over a one-year period. However, its bioactivity decreased from 50 to 80% after γ-irradiation at 25 and 50 kGy, respectively. Remarkably, the in vivo studies showed that the amount of new bone tissue formation induced by BMP-2 contained in PEM-coated Ti implants was not affected after air-drying of the implants and sterilization at 25 kGy indicating the full preservation of the growth factor bioactivity. Altogether, our results provided evidence of the remarkable property of PEM film coatings that both sequester BMP-2 and preserve its full in vivo osteoinductive potential upon both storage and γ-sterilization. The protective effects of PEM films on the growth factor bioactivity may be attributed to both the high water content in (PLL/HA) films (∼90%) and to their porosity, which may provide a “protein-friendly” environment similar to the natural extracellular matrix. This novel “off-the-shelf” technology of functionalised implants opens promising applications in prosthetic and tissue engineering fields

Summary. Both endogenous lubricin and injectable hyaluronic acid reduced cartilage friction coefficients, but by distinct mechanisms. Lubricin operated in boundary mode and hyaluronic acid shifted lubrication to mixed or hydrodynamic mode. Introduction. Intra-articular injections of viscous agents and boundary lubricants have been presented as options to mitigate the progression of articular cartilage damage after the onset of osteoarthritis. 1,2. Mechanically, these injections are predicted to lower the friction coefficient within a load bearing joint and consequently slow the propagation of damage at the articular surface. Tribologically, boundary lubricants and viscous agents are hypothesised to be effective through different mechanisms affecting boundary-mode lubrication and transition to mixed-mode lubrication, respectively. By normalizing sliding speeds on a Stribeck curve, this study evaluated the efficacy of injectable hyaluronic acid (HA) supplements and endogenous lubricin to alter tribological properties. Methods. Cartilage samples were extracted from the patellofemoral groove of neonatal bovine. A custom-built tribometer was used to measure friction coefficients of cartilage sliding against polished glass while in a lubricant solution. Cartilage samples were compressed to 20% strain and the normal load was allowed to reach a steady-state value before sliding at speeds from 0.1 to 10mm/s. For some samples, endogenous lubricin was removed from the surface as described previously. 3. via incubation in 1.5M NaCl in PBS for 20 minutes followed by re-equilibration in PBS for 1hr. Samples were tested in bathing solutions of PBS (control), equine synovial fluid (ESF), 10mg/ml HA, and a hydrophobic HA derivative (HYADD). Results. Friction coefficients as a function of sliding speed for some lubricants are presented. Comparisons show that lubricin removal from the tissue surface increases friction coefficients when PBS is used as a lubricant (p<0.05). At slow sliding speeds there was no significant difference between PBS and 10mg/ml HA, but at higher speeds HA transitions to a reduced friction coefficient. The hydrophobic HA (HYADD) provides significantly reduced friction coefficients compared to regular HA for speeds up to 7mm/s. These trends can be explained mechanically by normalizing data to a Hersey number (sliding speed∗viscosity/normal load). The data curves are similar to Stribeck curves which are characterised by different lubrication modes: boundary, mixed, and hydrodynamic. The PBS data appear to be in boundary mode (characteristic of highest friction coefficients), and a transition to mixed mode lubrication appears to occur within the ESF and HA solutions. The most viscous forms of HA (HYADD) lowered the friction coefficient to ≤ 0.05, with an apparent minimum at a Hersey number of ∼10. −6. m, suggesting that this lubricant may have enabled hydrodynamic lubrication, a phenomenon not noted previously in this system. Lubricin removal increased friction coefficient from 0.16 to 0.25, occurring at slow sliding speeds and Hersey numbers of 10. −12. m. Conclusions. Endogenous lubricin and injectable hyaluronan both effectively lower friction coefficients, but do so by distinct mechanism. At the same operating conditions (normal loads and sliding speed), lubricin lowers boundary mode friction coefficient, while hyaluronan shifts behavior to mixed mode (HA) or hydrodynamic mode (HYADD). The combination of the presence of lubricin on cartilage surface in the most viscous formulation of HA (HYADD) lowered the friction coefficient of articular cartilage from 0.26 to 0.05