There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels. The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders

Intervertebral disc (IVD) degeneration is the most frequent cause of Low Back Pain (LBP) affecting nearly 80% of the population [1]. Current treatments fail to restore a functional IVD or to provide a long-term solution, so, there is an urgent need for novel therapeutic strategies. We have defined the IVD extracellular matrix (ECM) profile, showing that the pro-regenerative molecules Collagen type XII and XIV, are uniquely expressed during fetal stages [2]. Now we propose the first fetal injectable biomaterial to regenerate the IVD. Fetal decellularized IVD scaffolds were recellularized with adult IVD cells and further implanted in vivo to evaluate their anti-angiogenic potential. Young decellularized IVD scaffolds were used as controls. Finally, a large scale protocol to produce a stable, biocompatible and easily injectable fetal IVD-based hydrogel was developed. Fetal scaffolds were more effective at promoting Aggrecan and Collagen type II expression by IVD cells. In a Chorioallantoid membrane assay, only fetal matrices showed an anti-angiogenic potential. The same was observed in vivo when the angiogenesis was induced by human NP cells. In this context, human NP cells were more effective in GAG synthesis within a fetal microenvironment. Vaccum-assisted perfusion decellularized IVDs were obtained, with high DNA removal and sGAG retention. Hydrogel pre-solution passed through 21-30G needles. IVD cells seeded on the hydrogels initially decreased metabolic activity, but increased up to 70% at day 7, while LDH assay revealed cytotoxicity always below 30%. This study will open new avenues for the establishment of a disruptive treatment for IVD degeneration with a positive impact on the angiogenesis associated with LBP, and on the improvement of patients’ quality of life. Acknowledgements: Financial support was obtained from EUROSPINE, ON Foundation and FCT (Fundação para a Ciência e a Tecnologia)

Introduction and Objective. Achilles tendon defect is difficult problem for orthopedic surgeon, and therefore the development of new treatments is desirable. Platelet-rich fibrin (PRF), dense fibrin scaffold composed of a fibrin matrix containing many growth factors, is recently used as regenerative medicine preparation. However, few data are available on the usefulness of PRF on Achilles tendon healing after injury. The objective of this study is to examine whether PRF promotes the healing of Achilles tendon defect in vivo and evaluated the effects of PRF on tenocytes in vitro. Materials and Methods. PRF were prepared from rats according to international guidelines on the literature. To create rat model for Achilles tendon defect, a 4-mm portion of the right Achilles tendon was completely resected, and PRF was placed into the gap in PRF group before sewing the gap with nylon sutures. To assess the histological healing of Achilles tendon defect, Bonar score was calculated using HE, Alcian-blue, and Picosirius-red staining section. Basso, Beattie, Bresnahan (BBB) score was used for the evaluation of motor functional recovery. Biomechanical properties including failure tensile load, ultimate tensile stress, breaking elongation, and elastic modulus were measured. We examined the effects of PRF on tenocytes isolated from rat Achilles tendon in vitro. The number of viable cells were measured by MTS assay, and immunostaining of ki-67 was used for detection of proliferative cells. Migration of tenocytes was evaluated by wound closure assay. Protein or gene expression level of extracellular matrix protein, such as collagen, were evaluated by immunoblotting, immunofluorescence, or PCR. Phosphorylation level of AKT, FGF receptor, or SMAD3 was determined by western blotting. Inhibitory experiments were performed using MK-2206 (AKT inhibitor), FIIN-2 (FGFR inhibitor), SB-431542 (TGF-B receptor inhibitor), or SIS3 (SMAD3 inhibitor). All p values presented are two-sided and p values < 0.05 were considered statistically significant. Results. In rat Achilles tendon defects, Bonar score was significantly improved in PRF group compared to control group. Collagen deposition at the site of Achilles tendon defect was observed earlier in PRF group. Consistent with the histological findings, BBB score was significantly improved in PRF group. PRF also significantly improved the biomechanical properties of injured Achilles tendon. Furthermore, proliferating tenocytes, labelled by ki-67 were significantly increased in PRF group. These data suggested PRF prompted the healing of Achilles tendon defect. Thus, we further examined the effects of PRF on tenocytes in vitro. PRF significantly increased the number of viable cells, the proliferative cells labelled by ki-67, and migratory ability. Furthermore, PRF significantly increased the protein expression levels of collagen-I, collagen-III, α-SMA, and tenascin-C in tenocytes. Next, we examined the signalling pathway associated with PRF-induced proliferation of tenocytes. PRF increased the phosphorylation level and induced nuclear translocation of AKT, known as key regulator of cell survival. PRF also induced the phosphorylation of FGF receptor. Inhibition of AKT or FGF-receptor completely suppressed the positive effects of PRF on tenocytes. Furthermore, we found that inhibition of FGF receptor partially suppressed the phosphorylation of AKT by PRF. Thus, PRF induced the proliferation of tenocytes via FGFR/AKT axis. We further evaluated the signalling pathway associated with PRF-induced expression of extracellular matrix. PRF increased the phosphorylation levels of SMAD3 and induced nuclear translocation of SMAD3. Furthermore, inhibition of TGF-B receptor or SMAD3 suppressed increased expression level of extracellular matrix by PRF. Thus, PRF increased expression level of extracellular matrix protein via TGF-BR/SMAD3 axis. Conclusions. PRF promotes tendon healing of the Achilles tendon defect and recovery of exercise performance and biomechanical properties. PRF increases the proliferation ability or protein expression level of extracellular matrix protein in tenocytes via FGFR/AKT or TGF-βR/SMAD3 axis, respectively

Background. The different biodegradable local antibiotic delivery systems are widely used in recent years. The aim of this study was to evaluate the bactericidal activity antibiotic loaded PerOssal pellet in vitro and its effectiveness in the treatment of Staphylococcus aureus induced chronic osteomyelitis. Material and methods. MALDI-TOF have been applied to microbiological diagnosis in patient with osteomyelitis. In most cases, Staphylococcus aureus was isolated. In vitro Ceftriaxone-Loaded PerOssal pellet were placed in middle agar plate containing a stock strain of Staphylococcus aureus. Plates were incubated at 37ºC for 24 hours. The zones of bacterial inhibition were recorded after 24, 48 and 72 hours of incubation. In vivo evaluation was performed by prospectively studying of 21 patients with a clinically and bacteriologically diagnosed Staphylococcus aureus induced osteomyelitis. Mean age was 38±4,2(26 to 53)). After radical surgical debridement and ultrasound cavitation, the bone cavity was full filled with Perosal pellets loaded with different antibiotics depending from the antibiotic sensitivity test. Endpoints were the absence of clinical manifestation of infection or disease recurrence, no need for further surgery. Results. In vitro showed after 24 hrs inhibition zone was 4,2 х 4,9 cm, after 72 hrs the inhibition zone was increased till 7,6 х 8,4 cm. During the subsequent time, there were no changes. Results of the clinical study evidenced no signs of infection in 18 patients (86% (CI 69,8;100)) (p<0,05) at the follow up, while 3 (14%(CI 0;30,2)) (p<0,05) subjects showed infection recurrence at 6 months from operation and 2 of them needed further surgical procedures. Conclusion. PerOssal as an antibiotic carrier stabilizes the action of the antibiotic. This antibiotic carrier system allows to choose an antibiotic individually for each patient according to the antibiotic sensitivity test and can be successfully used in clinical cases of osteomyelitis

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

Summary Statement. To test regenerative therapies for the intervertebral disc it is necessary to create a cavity in the nucleus polposus mantaining the annulus fibrosus intact. The transpedicular mechanical nucleotomy represents the best method for this purpose. Introduction. New cells/hydrogel based treatments for intervertebral disc (IVD) regeneration need to be tested on animal models before clinical translation. Ovine IVD represents a good model but doesn't allow the injection of a significant volume into intact IVD. The objective of the study was to compare different methods to create a cavity into ovine nucleus pulposus (NP) by enzymatic digestion (E), mechanical discectomy (M) and a combination of both (E+M), as a model to study IVD regeneration strategies with intact anulus fibrosus (AF). Methods. Ovine lumbar functional spinal units (FSU) were used. The transpedicular approach via the endplate route (2mm tunnel) was performed to access the NP with AF intact. FSUs were treated through M (Arthroscopic shaver), E (Trypsin/Collagenase) and E+M. The cavity was macro- and micro-scopically evaluated. The degradation of GAG (gel chromatography) around the cavity (inner AF) was assessed. The cavity volume was quantified through µCT after injection of Agarose gel/Contrast agent. Results. The cavity has been successfully created using all methods. The M group showed high reproducibility, low GAG degradation and no endplate thinning compared to other groups. The histology analysis demonstrated NP matrix degradation in E groups while the proteoglycan content was still homogenous in the M. The percentage of the cavity volume normalised to the total IVD volume was 5.2% ± 1.6 in E, 5% ±1.4 in E+M and 4, 2% ± 0.1in M. Discussion. M represents the best method to create a reproducible and less destructive cavity in the NP. Indeed, E-based methods perform better in terms of cavity volume but the GAG of the surrounding tissue may be affected. While a lesion of the end-plate might lead to further IVD degeneration, this approach is minimal invasive (2mm) and can be easily sealed using bone cylinder, cements or scaffolds. The biomechanical characterization and in vivo evaluation are on going

Summary Statement. The deletion of gangliosides enhanced OA development by elevating MMP-13 and ADAMTS-5 expression and accelerating chondrocyte apoptosis. Gangliosides possibly play suppressive roles in IL-1α-induced inflammatory signaling cascades. Introduction. We have previously reported that glycosphingolipids (GSLs) play chondroprotective roles in the cartilage degradation process [1]. Gangliosides, one of the series of GSLs, are known to be important in intercellular signal transduction and cell-to-cell recognition [2]. Therefore, we hypothesised that gangliosides are important in cartilage metabolisms among the GSLs species. The purpose of this study was to determine the functional role of gangliosides in the development of OA in murine models. Materials & Methods. We adopted systemic GM3 synthase deficient mice (GM3S. −/−. ) which lack most of the gangliosides [3], and wild-type C57BL/6 mice as controls (WT). We applied instability-induced OA model (transecting the medial collateral ligament and removing the cranial half of the medial meniscus [4]) and age-associated OA model (following up to 15 months) with these mice. We also applied IL-1α-induced OA model with femoral head cartilage explants ex vivo. Histological evaluation and quantification of released proteoglycan (PG), secreted MMP-13, and NO in the cultured media were performed. In vitro experiments with chondrocytes extracted from articular cartilages of both genotypes (GM3S. −/−. , WT) were also performed to check the mRNA expression of cartilage degrading enzymes (MMP-13, ADAMTS-5). To test the functional roles of gangliosides, transient GM3S transfection was applied to WT chondrocytes and quantification of MMP-13 and ADAMTS-5 expression was performed. Results. Both age-associated and instability-induced OA models showed cartilage degradation in GM3S. −/−. mice were significantly more severe than WT mice. The results of IL-1α-induced OA model showed gangliosides deletion enhanced chondrocyte apoptosis and accelerated cartilage degradation. Femoral heads from GM3S. −/−. showed significantly higher concentration of MMP-13 and NO in the cultured media than those from WT. In vitro experiments revealed that ganglioside deletion enhanced MMP-13 and ADAMTS-5 expression in the chondrocytes stimulated with IL-1α. The expression of these enzymes was significantly suppressed by overexpressed GM3S in WT chondrocytes. Discussion/Conclusion. The deletion of gangliosides enhanced OA development by elevating MMP-13 and ADAMTS-5 expression and accelerating chondrocyte apoptosis. The results of this study raised the possibility that gangliosides, synthesised mainly from GM3, would play crucial roles in maintaining cartilage homeostasis among the GSLs species. Moreover, the result of overexpression experiment indicated that gangliosides play suppressive roles in IL-1α-triggered inflammatory signaling cascades. Although further studies are required to confirm our speculation, gangliosides may be the target molecules of a novel and effective strategy for the treatment of OA

Objectives

To compare the therapeutic potential of tissue-engineered constructs (TECs) combining mesenchymal stem cells (MSCs) and coral granules from either Acropora or Porites to repair large bone defects.

Objectives

After an injury, the biological reattachment of tendon to bone is a challenge because healing takes place between a soft (tendon) and a hard (bone) tissue. Even after healing, the transition zone in the enthesis is not completely regenerated, making it susceptible to re-injury. In this study, we aimed to regenerate Achilles tendon entheses (ATEs) in wounded rats using a combination of kartogenin (KGN) and platelet-rich plasma (PRP).