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

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

The purpose of this study was to examine the effects of hyaluronic acid supplementation on chondrocyte metabolism in vitro. The clinical benefits of intra-articular hyaluronic acid injections are thought to occur through improved joint lubrication. Recent findings have shown that exogenous hyaluronic acid is incorporated into articular cartilage where it may have a direct biological effect on chondrocytes through CD44 receptors. Bovine articular chondrocytes were isolated and seeded into alginate constructs. These were cultured in medium containing hyaluronic acid at varying concentrations. Samples were assayed for biochemical and histological changes. There was a dose-dependent response to the exposure of hyaluronic acid to bovine articular chondrocytes in vitro. Low concentrations of hyaluronic acid (0.1 mg/mL and 1 mg/mL) significantly increase DNA, sulphated glycosaminoglycan and hydroxyproline synthesis. Immunohistology confirmed the maintenance of cell phenotype with increased matrix deposition of chondroitin-6-sulphate and collagen type II. These findings confirm a stimulatory effect of hyaluronic acid on chondrocyte metabolism

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

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

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

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

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

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

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

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

We have investigated the effects of the intra-operative application of a combination of hyaluronic acid and amniotic membrane on adhesions in the flexor tendons of a chicken model. We used 144 tendons which were partially divided and then repaired by a modified Kessler technique. There were four test groups: group 1, simple tendon repair, group 2, repair site wrapped with amniotic membrane, group 3, hyaluronic acid injected around the repair site, and group 4, repair site wrapped with amniotic membrane and hyaluronic acid injected within it. At three and six weeks, the extent of the adhesions and the healing of the tendon were evaluated macroscopically and histologically. The range of movement of the toe and tensile strength of the repaired tendons were measured at 20 weeks. The least adhesions were observed in group 4 but no significant difference was found in the healing of the tendons. Overall, the intra-operative application of a combination of hyaluronic acid and amniotic membrane appears to be effective in preventing adhesions of the flexor tendon

Osteoarthritis (OA) is the most common arthritis. Early OA is treated with pain-relieving medication while advanced diseases are treated with joint replacement. Intraarticular (IA) injection has been also used as a local therapy for OA. Only corticosteroids and hyaluronic acid has been clinically used for IA injection up to now. While these drugs are effective in alleviating pain relief and mitigating inflammation, they do not regenerate damaged cartilage. We have developed drug delivery system for OA treatment using a new molecule kartogenin which are known to have regenerative effects for cartilage. These systems include kartogenin-conjugated chitosan nano/microparticles, thermoresponsive nanospheres containing kartogenin and diclofenac, hyaluronic acid hydrogel containing PEGylated kartogenin micelles. We have found that injection of these systems arrested the progression of OA as well as inhibiting inflammation in surgically-induced OA model in rats. These data will be introduced in this talk

Articulating cartilage experiences a multitude of biophysical cues. Due to its primary function in distributing load with near frictionless articulation, it is clear that a major stimulus for cartilage homeostasis and regeneration is the mechanical load it experiences on a daily basis. While these effects are considered when performing in vivo studies, in vitro studies are still largely performed under static conditions. Therefore, an increasing complexity of in vitro culture models is required, with the ultimate aim to recreate the articulating joint as accurately as possible. We have for many years utilized a complex multiaxial load bioreactor capable of applying tightly regulated compression and shear loading protocols. Using this bioreactor, we have been able to demonstrate the mechanical induction of human bone marrow stromal cell (BMSC) chondrogenesis in the absence of exogenous growth factors. Building on previous bioreactor studies that demonstrated the mechanical activation of endogenous TGFβ, and subsequent chondrogenesis of human bone marrow derived MSCs, we have been further increasing the complexity of in vitro models. For example, the addition of high molecular weight hyaluronic acid, a component of synovial fluid, culture medium leads to reduced hypertrophy and increased glycosaminoglycan deposition. The ultimate aim of all of these endeavors is to identify promising materials and therapies during in vitro/ ex vivo studies, therefore reducing the numbers or candidates that are finally tested using in vivo studies. This 3R approach can improve the opportunities for success while leading to more ethically acceptable product development pathways

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

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

The fixation of articular fractures, with many small osteochondral fragments, is a challenging unmet need where a bone adhesive would be a useful adjunct to standard treatments. Whilst there are no such adhesives in current clinical use, preclinical animal models have demonstrated good healing of bone in unloaded models using an adhesive based on phosphoserine modified calcium phosphate cement (PM-CPC). An ex-vivo human bone core model has shown that this adhesive bonds freshly harvested human bone. To confirm this adhesive is capable of supporting loaded osteochondral fragments a porcine model has been developed initially ex-vivo on the path to an in-vivo study. In this model bone cores, harvested from the medial knee condyle, are glued in place with the adhesive. In-vivo adjacent pairs of bone cores would be replaced with adhesive and a control with conventional pin fixation respectively. As osteochondral bone fragments have both bone and cartilage components, this suggested a dual adhesive strategy in which components designed for each tissue type are used. This concept has been explored in an ex-vivo porcine pilot study presented herewith. At the subchondral bone level, the PM-CPC was used. At the cartilage level, a second adhesive, a methacrylated phosphoserine containing hyaluronic acid (MePHa) hydrogel designed specifically for soft tissues was applied. This is a challenging model as both adhesives have to be used simultaneously in a wet field. The pilot showed that once the subchondral component is glued in place, the PM-CPC adhesive intruding into the cartilage gap can be removed before applying the cartilage adhesive. This enabled the MePHa adhesive to be injected between the cut cartilage edges and subsequently light-cured. This two-stage gluing method is demanding and an in-vivo pilot is necessary to perfect and prove the operative technique. Acknowledgements: The human bone core project was partially financed by Innovation Fund of Västra Götaland Region, Sweden. The MePHa hydrogel work was supported by a Swiss National Fund grant # CR23I3_159301

Biomaterials with mechanical or biological competence are ubiquitous in musculoskeletal disorders, and understanding the inflammatory response they trigger is key to guide tissue regeneration. While macrophage role has been widely investigated, immune response is regulated by other immune cells, including neutrophils, the most abundant leukocyte in human blood. As first responders to injury, infection or material implantation, neutrophils recruit other immune cells, and therefore influence the onset and resolution of chronic inflammation, and macrophage polarization. This response depends on the physical and chemical properties of the biomaterials, among other factors. In this study we report an in vitro culture model to describe the most important neutrophil functions in relation to tissue repair. We identified neutrophil survival and death, neutrophils extracellular trap formation, release of reactive oxygen species and degranulation with cytokines release as key functions and introduced a corresponding array of assays. These tests were suitable to identify clear differences in the response by neutrophils that were cultured on material of different origin, stiffness and chemical composition. Overall, substrates from biopolymers of natural origin resulted in increased survival, less neutrophil extracellular trap formation, and more reactive oxygen species production than synthetic polymers. Within the range of mechanical properties explored (storage modulus below 5 k Pa), storage modulus of covalently crosslinked hyaluronic acid hydrogels did not significantly alter neutrophils response, whereas polyvinyl alcohol gels of matching mechanical properties displayed a response indicating increased activation. Additionally, we present the effect of material stiffness, charge, coating and culture conditions in the measured neutrophils response. Further studies are needed to correlate the neutrophil response to tissue healing. By deciphering how neutrophils initiate and modulate the immune response to material implantation, we aim at introducing new principles to design immunomodulatory biomaterials for musculoskeletal disorders. Acknowledgments. This work was supported by the AO Foundation, AO CMF, grant AOCMF-21-04S

INTRODUCTION. Intervertebral disc (IVD) degeneration is not completely understood because of the lack of relevant models. In vivo models are inappropriate because animals are quadrupeds. IVD is composed of the Nucleus Pulposus (NP) and the Annulus Fibrosus (AF), an elastic tissue that surrounds NP. AF consists of concentric lamellae made of collagen I and glycosaminoglycans with fibroblast-like cells located between layers. In this study, we aimed to develop a novel 3D in vitro model of Annulus Fibrosus to study its degeneration. For this purpose, we reproduced the microenvironment of AF cells using 3D printing. METHOD. An ink consisting of dense collagen (30 mg.mL. -1. ) and tyramine-functionalized hyaluronic acid (THA) at 7.5 mg.mL. -1. was first designed by modulating pH and [NaCl] in order to inhibit the formation of polyionic complexes between collagen and THA. Then, composite inks were printed in different gelling baths to form collagen hydrogels. Last, THA photocrosslinking using eosin and green light was performed to strengthen hydrogels. Selected 3D printed constructs were then cellularized with fibroblasts. RESULTS. The physicochemical study revealed that collagen/THA solutions (4:1 ratio) used at pH 5 with 200 mM NaCl were homogenous. In addition, collagen fibrils were observed in these solutions. The dense composite collagen/THA inks printed in a 2X PBS bath rapidly gelled and the photo-crosslinking increased the mechanical properties by 2 to reach 25 kPa (Young's modulus). Then, 3D printing parameters were optimized (85 kPa, extrusion, 4.5 mm/s speed and 80% fill-in percentage) to generate flat and anisotropic lamellae observed by polarized light microscopy. For the in vitro study, several anisotropic layers were printed and fibroblasts seeded between them. Cells adhered to layers, spread, proliferate and aligned along the axis of printed layers. CONCLUSION. Taken together, these results show it is possible to reproduce in vitro the main AF's biochemical and physical properties

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