Hip Osteoarthritis (HOA) is the most common joint disorder and a major cause of disability in the adult population, leading to total hip replacement (THR). Recently, evidence has mounted for a prominent etiologic role of femoroacetabular impingement (FAI) in the development of early OA in the non-dysplastic hip. FAI is a pathological mechanical process, caused by abnormalities of the acetabulum and/or femur leading to damage the soft tissue structures. FAI can determine chondro-labral damage and groin pain in young adults and can accelerate HOA progression in middle-aged adults.

The aim of the study was to determine if the presence of calcium crystal in synovial fluid (SF) at the time of FAI surgery affects the clinical outcomes to be used as diagnostic and predictive biomarker.

49 patients with FAI undergoing arthroscopy were enrolled after providing informed consent; 37 SFs were collected by arthrocentesis at the time of surgery and 35 analyzed (66% males), median age 35 years with standard deviation (SD) 9.7 and body mass index (BMI) 23.4 kg/m²; e SD 3.

At the time of surgery, chondral pathology using the Outerbridge score, labral pathology and macroscopic synovial pathology based on direct arthroscopic visualization were evaluated. Physical examination and clinical assessment using the Hip disability & Osteoarthritis Outcome Score (HOOS) were performed at the time of surgery and at 6 months of follow up. As positive controls of OA signs, SF samples were also collected from cohort of 15 patients with HOA undergoing THR and 12 were analysed.

45% FAI patients showed CAM deformity; 88% presented labral lesion or instability and 68% radiographic labral calcification. 4 patients out of 35 showed moderate radiographic signs of OA (Kellegren-Lawrence score = 3). Pre-operative HOOS median value was 61.3% (68.10-40.03) with interquartile range (IQR) of 75-25% and post-operative HOOS median value 90% with IQR 93.8-80.60. In both FAI and OA patients the calcium crystal level in SFs negatively correlated with glycosaminoglycan (component of the extracellular matrix) released, which is a marker of cartilage damage (Spearman rho=-0.601, p<0.001).

In FAI patients a worst articular function after surgery, measured with the HOOS questionnaire, was associated with both acetabular and femoral chondropathy and degenerative labral lesion. Moreover, radiographic labral calcification was also significantly associated with pain, worst articular function and labral lesion. Calcium crystal level in SFs was associated with labral lesions and OA signs.

We concluded that the levels of calcium crystals in FAI patients are correlated with joint damage, OA signs and worst post-operative outcome. The presence of calcium crystals in SF of FAI patients might be a potential new biomarker that might help clinicians to make an early diagnosis, evaluate disease progression and monitor treatment response.

Objectives

Meniscal injuries are often associated with an active lifestyle. The damage of meniscal tissue puts young patients at higher risk of undergoing meniscal surgery and, therefore, at higher risk of osteoarthritis. In this study, we undertook proof-of-concept research to develop a cellularized human meniscus by using 3D bioprinting technology.

Osteoarthritis (OA), the most common chronic degenerative joint disease, is characterized by inflammation, degradation of the articular cartilage and subchondral bone lesions, causing pain and decreased functionality.

NF-κB pathway is involved in OA and, in most cases, its activation depends on the phosphorylation and degradation of IκBα, the NF-κB endogenous inhibitor that sequesters NF-κB in the cytosol. Under inflammatory stimuli, IκBα is degraded by the IKK signalosome and NF-κB moves into the nucleus, inducing the transcription of inflammatory mediator genes and catabolic enzymes. The IKK signalosome includes IKKβ and IKKα kinases, the latter shown to be pivotal in the OA extracellular matrix derangement. The current OA therapies are not curative and nowadays, the preclinical research is evaluating new structure-modifying pharmacological treatments, able to prevent or delay cartilage degradation.

N-acetyl phenylalanine derivative (NAPA), is a derivative of glucosamine, a constituent of the glycosaminoglycans of cartilage and a chondroprotective agent. Previous in vitro studies showed the ability of NAPA to increase cartilage components and to reduce inflammatory cytokines, inhibiting IKKα kinase activity and its nuclear migration.

The present study aims to further clarify the effect of NAPA in counteracting OA progression, in an in vivo mouse model after destabilization of the medial meniscus (DMM).

Mice were divided into 3 groups:

-

DMM group: DMM surgery without NAPA;

DMM surgery was performed in the right knee, according to Glasson SS [2], while the left knee did not undergo any surgery. Four weeks after surgery (mild-to-moderate OA), some animals received one intra-articular injection of NAPA (2.5 mM) and after 2 weeks, the animals were pharmacologically euthanized. The mice of the 1^st group were euthanized 4 weeks after DMM and those of the 3^rd group after 6 weeks from their arrival in the animal facility. At the end of experimental times, both knee joints of the animals were analyzed through histology, histomorphometry, immunohistochemistry and subchondral bone microhardness.

The injection of NAPA significantly improved cartilage structure, increased cartilage thickness (p<0.0005), reduced Chambers and Mankin scores (p<0.005), fibrillation index (p<0.005) and decreased MMP13 (p<0.05) and ADAMTS5, MMP10, and IKKα (p<0.0005) staining. The microhardness measurements did not shown statistically significant differences between groups.

This study demonstrated the chondroprotective activities exerted by NAPA in vivo. NAPA markedly improved the physical structure of articular cartilage and reduced the amount of catabolic enzymes, and therefore of extracellular matrix remodeling. The reduction in OA grading and catabolic enzymes paralleled the reduction of IKKα expression. This further hints at a pivotal role of IKKα in OA development by regulating MMP activity through the control of procollagenase (MMP10) expression. We believe that the preliminary preclinical data, here presented, contribute to improve the knowledge on the development of disease modifying drugs since we showed the ability of NAPA of reverting the surgically induced OA in the widely accepted DMM model.

In clinical orthopedics suitable materials that induce and restore biological functions together with the right mechanical properties are particularly needed for the regeneration of musculoskeletal tissue. An innovative solution to answer this need is represented by tissue engineering. This technique could overcome the limits of traditional approaches involving the use of homologous, autologous or allogenetic tissue (e.g. tissue availability, immune rejection and pathogen transfer). In this field, rapid prototyping techniques are emerging as the most promising tool to realize three-dimensional tissue constructs with highly complex geometries.

Based on CAD/CAM technology, rapid prototyping allows development of patient-specific 3D scaffolds from digital data obtained with latest generation imaging tools. These structures can be realized in different materials, tailoring their mechanical properties and architectural features. Most rapid prototyping techniques allow the creation of acellular 3D scaffolds, which must be subsequently seeded with cells. Conversely, 3D bioprinting can deposit bio-ink containing molecules/cells, providing desired spatial distribution of growth factors/cells within the scaffold. The need of printable materials suitable for processing with inkjet, dispensing, or laser-print technologies, forces the use of matrices within a specific range of viscosity. However, these materials have low mechanical features. To overcome this problem and to obtain a final construct with good mechanical properties, bioprinting tissue fabrication can rely on the alternate deposition of thermoplastic materials and cell-laden hydrogels. Since mechanical performance is determined not only by the material properties but also by the geometry (microarchitecture) of the structure, printing parameters can be modified to obtain the desired features.

The new 3D platform available at Rizzoli Orthopaedic Institute, consisting of a Computer Tomography (GE Medical Systems, Milano, Italia) and a 3D Bio-Printer (RegenHU, Villaz-St-Pierre, Switzerland) is used to address the above-mentioned issues. Preliminary results showed that it is possible to modify the microarchitecture of the printed structures adjusting their apparent density and stiffness in the range of the trabecular bone tissue. Additionally, it has been proven that the calcium phosphate based paste, used as bioink, allows cell attachment and proliferation. Therefore, the platform allows to print scaffolds with open and interconnected porosities and suitable mechanical properties. They can be filled with different components such as cells or soluble growth factors at specific locations.

INTRODUCTION

Fresh bipolar shell osteochondral allograft (FBOA) is a controversial treatment option for post-traumatic ankle arthritis. Immunological response to transplanted cartilage may play a role in failure. Aim of the study is to compare two groups of patients who received FBOA in association or not to immunosuppressive therapy.

Objective: Different

Methods: have been proposed to date to achieve the regeneration of hyaline cartilage in osteochondral lesions of the talus (OLT). The aim of this study was to present a new one-step arthroscopic procedure with the use of mesenchimal stem cells (MSC) supported on a collagen scaffold and Platelet Rich Fibrin (PRF).

Methods: 14 patients with a diagnosis of OLT underwent this procedure. The MSC were harvested from the posterior iliac crest and concentrated directly in the operating room. An ankle arthroscopy was performed with lesion detection and curettage. The cell concentrate was mixed with a collagen paste as scaffold and with PRF as a pool of growth factors in order to have a final composite to fill the lesion site. Partial weight bearing for 2 months and early ROM was advised postoperatively.

Results: According to the American Orthopaedic Foot and Ankle Score (AOFAS) system the patients had a preoperative score of 65.1 (range 35–79), a postoperative of 69.4 (range 61–97) at 6 months and of 83.6 (range 65–100) at 12 months follow up. MRI control at 6 and 12 months showed a progression of the reparative process in the osteochondral lesions. Histological and immuno-hystochemical analysis on a sample biopsed during a control arthroscopy at 12 months confirmed the hyaline quality of the regenerated cartilage.

Conclusions: This one-step technique demonstrated to be capable to regenerate hyaline cartilage, with the advantages of a reduced surgical time, lower costs and lower patient’s morbidity.

Introduction Osteochondral lesions of the talus are a common occurrence especially in sports injuries. The biomechanical nature of the ankle joint makes it susceptible to sprains which can cause damage not only to the capsulo-ligamentous structures, but also to the joint cartilage and subchondral bone. As it is known, joint cartilage is a highly specialized and multitask tissue. Because joint cartilage has poor reparative capability, damage may be irreversible and as a consequence, can also lead to osteoarthritis. The purpose of this study is to review the results of a series of patients treated with autologous chondrocytes implantation (A.C.I.) and to describe the evolution in surgical technique that we have been implemented in the last 8 years.

Methods Thirty-nine patients with a mean age of 27 8 years affected by osteochondral lesions of the talus > 1.5 cm2, were treated by autologous chondrocyte implantation. All patients were checked clinically and by MRI up to 4 years follow-up. The first 9 patients received the ACI by open technique and the remaining 30, arthroscopically. In the last 10 patients the cartilage harvested from the detached osteochondral fragment was used for the colture. All patients were checked clinically (AOFAS score), radiographically and by MRI, before surgery, at 12 months and at follow-up. Eleven patients underwent a second arthroscopy with a bioptic cartilage harvest at 1 year follow-up. Samples were stained with Safranin-O and Alcian Blue. Immunohistochemical analysis for collagen type II was also performed

Results Before surgery the mean score was 48.4 17 points, at 12 months 90.9 12 (p< 0.0005), while at follow up was 93.8 8 (p< 0.0005) demonstrating an improvement over time. The histological and immunohistological analyses performed on the cartilage samples using Safranin-O, Alcian Blue staining and anti-human collagen type II antibody respectively showed a typical cartilage morphology, were positive for collagen type II and for proteoglycans expression.

Conclusions The clinical and histological results have confirmed the validity of the technique utilized, with laboratory data confirming the newly formed cartilage was of hyaline type for all the cases evaluated.

Aim of study: The development of tissue engineering techniques evidenced that the healing of injured ligaments require the interactions of different cell types, local cellular environment and the use of devices. In order to gain new information on the complex interactions between mesenchymal stem cells (MSCs) and biodegradable scaffold, we analysed in vitro the proliferation, vitality and phenotype of MSCs grown onto a multilayered-woven-cylindric-array of Hyaff-11A8 fiber configured as ligament scaffold.

Methods: Sheep MSCs were isolated from bone marrow aspirates and grown at two different density (7,5x106/cm and 15x106/cm) in the scaffold. At different time points (2, 4, 6 days) cellular proliferation was analysed by MTT test and cellular viability by calcein-AM immunofluorescence dye and confocal microscopy analysis. Moreover, hyaluronic acid receptor (CD44) and typical matrix ligament proteins (collagen type I, III, laminin, fibronectin, actin) were evaluated by immunohistochemistry.

Results: MSCs growth was cell density-dependent and cells were uniformly distributed inside and along the scaffold. Confocal analysis showed that MSCs completely wrap the fibers at both cell concentrations analysed and were all viable both outside and inside the scaffolds only using the lower cell concentration. Moreover, MSCs expressed CD44, collagen type I, III, laminin, fibronectin and actin.

Conclusion: These data demonstrate that MSCs well survive in a hyaluronic acid-configured ligament scaffold expressing a protein important for scaffold interaction, like CD44, and proteins responsible of the functional characteristic of the ligaments.

Aims: The maintenance of the original phenotype by isolated chondrocytes grown in vitro is an important requisite for their use in repairing damaged articular cartilage. The methods to verify the expression of cartilage specific molecules usually involve destructive procedures to recover the cells from the scaffolds for tests. The aim of this study was to find a soluble marker able to attest the occurrence of a differentiation process by chondrocytes grown onto a biomaterial used for cell transplantation. We turned our attention to cathepsin B which is known to be abnormally synthesized in de-differentiated chondrocytes and scarcely produced in the differentiated ones.

Methods: The production of cathepsin B by human articular chondrocytes expanded in vitro and then grown onto a hyaluronan-based polymer derivative (Hyaff“-11) three-dimensional scaffold was evaluated with a specific ELISA and by immunohistochemical analysis at different experimental times (1hour, 1 day, 7, 14, 21 days) together with the expression of mRNA by Real Time PCR.

Results: Cathepsin B is always secreted by the cells grown onto the biomaterial but the protein levels increased from the first day after seeding up to 7 days (p< 0.01), then decreased progressively and significantly until day 21 (p< 0.01). The immunohistological data confirmed those obtained by the ELISA test. Cathepsin B staining was particularly evident at day 7 after cells were seeded onto the biomaterial, and then progressively decreased up to 21 days; at this experimental time point, the totality of cells were negative. Real-time PCR monitoring with the LightCycler using fluorescent dye allowed rapid and sensitive detection of cathepsin B mRNAs from the patient samples. The mRNA levels increased for up to 7 days of culture and slightly decreased until day 21. However, no significant differences were observed.

Conclusions: We can identify in cathepsin B a soluble marker of differentiated chondrocytes phenotype useful in the monitoring of autologous chondrocyte transplantation performed by means of different carriers. Its low concentration in the constructs culture medium could be indicative of a phenotypic stability. The introduction of mature cells inside the chondral defects could help to regenerate damaged hyaline articular cartilage better and faster.

The purpose of this study is to demonstrate the validity of the autologous chondrocytes transplantation (A.C.T.) technique implemented over the last 6 years in the treatment of osteochondral lesions of the talus.

Our case study included 22 patients (12 males and 10 females), with an average age of 27 years affected by osteochondral lesions of the talus surface. All lesions were > 1.5 cm2, monofocal, and post-traumatic in origin. The first 9 patients received ACT (Genzyme technique) and the remaining 13 patients received ACT with an arthroscopic technique. In 6 of the patients, the cartilage harvested from the detached osteochondral fragment was used for culturing, avoiding the first step arthroscopy in the knee. Before surgery, all patients were assessed clinically, radiographically, and using MRIs. For clinical evaluation patients were assessed using the American Foot & Ankle Society 100 point score. Before surgery the mean score was 48.4 points. 11 patients underwent second-look arthroscopy at one year during which a biopsy was harvested for histologic analysis of the reconstructed cartilage. Of these, 9 patients (Genzyme technique) also had hardware removed.

The mean follow-up of the 22 patients was 36 months. At follow-up, all patients but one were satisfied with their results. With regards to the clinical results evaluated using the American Foot and Ankle Society score, an average of 90.5 was obtained at 24 months, while at 36 months the average score (19 patients) was 94.0 (range 54–100). During follow-up arthroscopy, 4 patients had mild fibrosis and 1 patient required regularization of flap overgrowth causing pain.

The clinical and histological results have confirmed the validity of the surgical technique utilized with no subjective nor objective complications. An improvement of the symptoms and of articular function has also been observed: laboratory data confirmed the histological appearance of the newly formed hyaline cartilage in all cases evaluated. Immunohistochemistry showed a positive staining for collagen type II located in the extracellular matrix and in the chondrocytes in the healthy and transplanted cartilage biopsies. All the specimens studied were also positive for proteoglycans expression as was the Alcian blue reaction, which highlighted the presence of these fundamental components of a cartilaginous matrix.