Cartilage Biopsy Research Articles

Intra-Articular Cartilage Calcification Associated with Type II Pro-Collagen Accumulation in Chondrocytes and Abnormal Fibrils in the Extra Cellular Matrix (ECM): Case Report

Objective: The purpose of this case-study was to perform morphological and molecular analysis of articular cartilage biopsies from the femoral condyle of a 33 year old woman with intra-articular calcification in the right knee joint and compare the findings with those of normal cartilage. Methods: Femural condyle cartilage biopsies were used for Light Microscopy (LM), Transmission Electron Microscopy (TEM), explant culturing and DNA mutation analysis of the COL2A1 gene. Results: X-ray of the affected knee joint showed intra-articular calcifications on the femur condyle, tibia and meniscus. Pathological LM and TEM examination of cartilage biopsies revealed calcified islands morphologically identical to calcium pyro-phosphate dehydrate (CPPD) and hydroxyapatite (HA)-like crystals. In addition, chondrocytes showed accumulation of pro-collagen molecules. With explant culturing and immunochemistry, it was confirmed that matrix calcification correlated with high intracellular matrix accumulation and expression of type X collagen. The induction of hypertrophy in chondrocytes was further associated with matrix vesicle (MV) release and a prominent calcification zone. Surprisingly, TEM showed crystal development on thick abnormal type II collagen fibrils suggesting that these ECM components might nucleate and contribute to calcification. Conclusions: We suggest that intra-articular calcification may be associated with type II pro-collagen accumulation in chondrocytes. In particular, we hypothesize that matrix accumulation may induce hypertrophy and type X collagen expression in cartilage cells and release of MV’s into the ECM, which together with thick abnormal type II collagen hetero-fibrils, are responsible for crystal deposition in the ECM.

Manganese dioxide nanoparticles modulate oxidative stress and protect cartilage from interleukin-1β induced degradation

Purpose: Osteoarthritis (OA) is associated with chronic joint inflammation, whereby pro-inflammatory cytokines such as IL-1β upregulate reactive oxygen species (ROS) production while downregulating anti-oxidants in cells. The resulting oxidative stress leads to extracellular matrix degradation, joint inflammation, and chondrocyte death and senescence. The overall goal of this research is to develop a bioactive nanoparticle (NP) system that scavenges ROS in cartilage to modulate the impact of joint inflammation. Manganese dioxide (MnO2) catalyzes the breakdown of hydrogen peroxide (H2O2) and is currently being evaluated for scavenging ROS in inflammatory diseases such as atherosclerosis. We previously reported engineered MnO2 NPs with properties that enable them to penetrate cartilage and become endocytosed by chondrocytes. The objective of this study was to evaluate the chondroprotective mechanisms of MnO2 NPs and their in vivo retention and biocompatibility. Methods: MnO2 NPs were synthesized and characterized as previously described. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2D culture and cartilage explants were confirmed by fluorescent imaging after 24 hours of incubation. Gene expression analysis of both catabolic and ROS mediator genes was conducted on a monolayer of primary bovine chondrocytes challenged by human recombinant IL-1β in the presence of MnO2 NPs after 24-hour culture. Fresh bovine cartilage biopsies were evaluated for loss of glycosaminoglycans (GAG), the production of nitric oxide (NO) and cell viability over a 14-day period when challenged by IL-1β in the presence of MnO2NPs. Articular joint retention of Alexa Fluor 750 and 594 conjugated NPs in Lewis rats was monitored using an in vivo IVIS Imaging System. Long-term cytocompatibility of the particles 6 weeks after intraarticular injection in healthy rat joints was assessed histologically using GEKO©, a tool for quantitative grading of toluidine stained knees. Results: The PEG stabilized MnO2 NPs had a hydrodynamic diameter of 15 nm, TEM size of 8 nm and had a cationic surface charge. The NPs effectively scavenged H2O2, with 5μg/mL MnO2 NPs neutralizing 55% of 100uM H2O2 in PBS. Chondrocytes in monolayer showed uptake of MnO2 NPs without cytotoxicity. Furthermore, the NPs reduced the gene expression of catabolic mediator, heme oxygenase-1, catabolic factor matrix metalloproteinase 3, antioxidant mediator nuclear factor erythroid 2-related factor 2, antioxidants catalase, glutathione peroxidase and manganese superoxide dismutase by chondrocytes (Fig. 1A) indicating protection of the particles against oxidative stress. When incubated with cartilage explants, the MnO2 NPs penetrated through the cartilage depth and were found in the extracellular matrix as well as intracellularly in chondrocytes. In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased GAG loss by 50% and NO production by 40% compared to controls (Fig. 1B and C). Importantly, treatment with MnO2 NPs improved viability of chondrocytes in cartilage explants over the 2-week study. Upon in vivo injection, the MnO2 NPs persisted in the joint space over one week, with 20% of the initial fluorescent signal still observed by day 13 (Fig. 2A and B). Biodistribution and histological analysis (Fig. 2C) revealed accumulation of particles at the chondral surfaces, and colocalization of the NPs with the lacunae of chondrocytes, indicating particle penetration into the cartilage and uptake by chondrocytes following intraarticular injection. A gradient of NPs existed in cartilage with the highest levels of NPs on the articular surface which decreased with tissue depth. Histological analysis also revealed no adverse effects of intra-articular MnO2 NP injection into healthy animals. Histological parameters such as synovium thickness, total cartilage degeneration width and cartilage matrix loss width were comparable in knees injected with the NPs and those injected with saline after 6 weeks, indicating biocompatibility of the particles in vivo (Fig. 2D). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. In vitro, the NPs protected chondrocytes in cartilage explants from IL-1β mediated oxidative stress and apoptosis. Further analysis of the joint confirmed penetration of MnO2 NPs into cartilage in vivo. Given their joint retention time and ROS scavenging capacity, these NPs could target oxidative stress to treat or prevent OA. As the NPs show intracellular localization in chondrocytes, they could also deliver other chondroprotective agents including nucleic acids to target multiple pathways in the OA pathology. Further studies will focus on the therapeutic impact of MnO2 NPs in OA disease models.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Longitudinal assessment of cartilage composition by high-field MRI in patients with low-grade knee cartilage injury

Purpose: If left untreated, early-stage articular cartilage lesions may progress to full-thickness, higher-grade lesions, possibly leading to further degeneration of the joint surface. This process is typically associated with the breakdown of extracellular matrix macromolecules, such as collagen type 2 and proteoglycans. This makes collagen and glycosaminoglycans (GAG) as biomarkers to monitor the status of the articular cartilage and for studies on cartilage repair. The aim of this study was to evaluate the sensitivity of a comprehensive Magnetic Resonance Imaging (MRI) approach in detecting changes in cartilage composition in ICRS grade I/II focal lesions over a 12-month period. Methods: MRI was performed at baseline, 8-day, 3, 6 and 12-month, both at 7T and 3T, on 20 subjects with ICRS Grade I/II cartilage defects. Collagen bi-layer organization was assessed by T2 mapping, and GAG content was measured both by 23Na and gagCEST-MRI (7T only) (Fig. 1). Regions-of-interest were defined in both defective and normal appearing cartilage (weight bearing [WBR] and non-weight bearing [NWBR] regions), and transferred to T2 maps, 23Na and gagCEST images for quantitative analysis. Patient functional status was assessed via the Knee injury and Osteoarthritis Outcome Score (KOOS) questionnaire. Results: Higher T2 values were measured at baseline in deep and superficial layers of the defective vs healthy WBR and NWBR, both at 3T and 7T. While T2 values remained unchanged at 7T, a T2 decrease was observed over 12 months at 3T in deep (-15%) and superficial (-17%) layers of the damaged cartilage. 23Na-MRI signal intensities were ∼25% lower in the defective area vs other regions, with no large variation over the 12-month follow-up. gagCEST measurements at 7T appeared in good agreement with 23Na-MRI results. KOOS values remained stable over the 12-month time period. Conclusions: These results point to a progressive alteration of collagen structure in the defective region (despite stable KOOS), without large changes in GAG levels over 1 year in this population. These data support the use of MRI as a noninvasive alternative to cartilage biopsy for the detection of subtle changes in cartilage composition.

Severe visual impairment and subclinical encephalitis preceding clinical signs of chondritis in relapsing polychondritis

AbstractA 71‐year‐old woman with a 6‐month history of relapsing bilateral anterior scleritis presented with severe right visual impairment due to posterior scleritis. Despite radiological signs of encephalitis, the patient and her family members noticed no cognitive decline. The patient subsequently developed slight auricular pain without any visual changes such as redness or swelling, which, however, showed increased uptake of 18F‐fluorodeoxyglucose on positron emission tomography. Auricular cartilage biopsy revealed perichondrial inflammation suggesting relapsing polychondritis. Steroid therapy improved her symptoms and radiological findings. This case illustrates that asymptomatic brain inflammatory lesions can precede clinical signs of chondritis in relapsing polychondritis, and that auricular cartilage biopsy should be considered even with mild auricular pain without apparent clinical findings of inflammation.

Manganese dioxide nanoparticles mitigate oxidative stress in osteoarthritic cartilage

Purpose: Osteoarthritis (OA) is associated with chronic joint inflammation, whereby pro-inflammatory cytokines such as IL-1β upregulate reactive oxygen species (ROS) production while downregulating anti-oxidants in cells. The resulting oxidative stress leads to extracellular matrix degradation, joint inflammation, and chondrocyte death and senescence. The role of oxidative stress in OA etiology makes it a therapeutic target to ameliorate some of the devastating symptoms of the disease. The overall goal of this research is to develop a bioactive nanoparticle (NP) system that scavenges ROS in cartilage to modulate the impact of joint inflammation. Manganese dioxide (MnO2) catalyzes the breakdown of hydrogen peroxide (H2O2) and is currently being evaluated for scavenging ROS in inflammatory diseases such as atherosclerosis. We hypothesize that MnO2 NPs will protect cartilage from inflammation-induced oxidative stress. To do this, we have engineered chondroprotective MnO2 NPs with properties that enable them to penetrate into the cartilage. Methods: MnO2 NPs were synthesized by the oxidation of potassium permanganate with polyallyamine hydrochloride and further stabilized with polyethylene glycol (PEG). The NPs were characterized for size and zeta potential in phosphate buffer saline (PBS) by Dynamic Light Scattering (DLS) and polydispersity by transmission electron microscopy (TEM). Scavenging capacity of MnO2 NPs was evaluated by a H2O2 colorimetric detection kit. Uptake of Alexa Fluor 488 conjugated NPs by bovine chondrocytes in 2-D culture and cartilage explants were evaluated by fluorescent imaging after 24 hours of incubation. The cytotoxicity of NPs to chondrocytes was tested using the MTS colorimetric assay. Fresh bovine cartilage biopsies were challenged with human recombinant IL-1β, to mimic OA-like cartilage, in the presence of MnO2 NPs and evaluated for loss of glycosaminoglycans (GAG), a key component of cartilage extracellular matrix, and production of nitric oxide (NO), a key reactive nitrogen species (RNS) related to oxidative stress in OA, over a 9-day period. The loss of GAG was quantified using spectrophotometric Dimethylmethylene Blue Assay while NO concentrations were evaluated by the Griess reaction. Articular joint retention of Alexa Fluor 750 conjugated NPs in rats was monitored using an in vivo IVIS Imaging System. Results: The PEG stabilized MnO2 NPs had a hydrodynamic size of 15 nm (number weighted) and TEM size of 8 nm. The NPs had neutral charge. The NPs effectively scavenged H2O2, with 5ug/mL MnO2 NPs neutralizing 55% of 100uM H2O2. Chondrocytes in monolayer showed uptake of MnO2 NPs, which escaped the endosomes and localized perinuclearly without cytotoxicity (Fig. 1). The MnO2 NPs penetrated through the depth of the cartilage biopsies and remained in the tissue matrix. Interestingly, the NPs were also endocytosed by resident chondrocytes (Fig. 2). In cytokine challenged cartilage explants, supplementation with MnO2 NPs decreased NO production by 80% and reduced GAG loss by 55% compared to controls (Fig. 3A and B). The MnO2 NPs after in vivo injection in articular joints displayed a linear decline (R2 = 0.933) in retention with time. Quantification of fluorescent signals showed that 78% of the initial amount of NPs remained in joints after 5 days (Fig. 4A and B). Conclusions: MnO2 NPs have the potential to reduce oxidative stress in osteoarthritic cartilage. Given their joint retention time and ROS scavenging capacity, these NPs could target oxidative stress to treat or prevent OA. As the NPs show intracellular localization in chondrocytes, they could also deliver other chondroprotective agents including nucleic acids to target multiple pathways in the OA pathology. Further studies will focus on the therapeutic impact of MnO2 NPs to joint tissues in vivo.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Evaluation of cartilage specific matrix synthesis of human articular chondrocytes after extended propagation on microcarriers by image analysis.

Cell-based technologies for the repair of cartilage defects usually rely on the expansion of low numbers of chondrocytes isolated from biopsies of healthy cartilage. Proliferating chondrocytes are known to undergo dedifferentiation characterized by downregulation of collagen type II and proteoglycan production, and by upregulation of collagen type I synthesis. Re-expression of cartilage specific matrix components by expanded chondrocytes is therefore critical for successful cartilage repair. Human articular chondrocytes were expanded on microcarriers Cytodex 3. The growth area was increased by adding empty microcarriers. Added microcarriers were colonized by bead-to-bead transfer of the cells. The chondrocytes were harvested from the microcarriers and characterized by their ability to synthesize collagen type II when cultivated in alginate beads using chondrogenic growth factors. A semi-automatic image analysis technique was developed to determine the fractions of collagen type II and type I positive cells. The expansion of human articular chondrocytes on microcarriers yielded high cell numbers and propagation rates compared to chondrocytes expanded in flask culture for one passage. The proportion of collagen type II positive cells compared to collagen type I synthesizing cells was increased compared to chondrocytes expanded using conventional methods. The matrix synthesis upon treatment with chondrogenic factors IGF-I and BMP-7 was enhanced whereas TGF-ss had an inhibitory effect on microcarrier expanded chondrocytes. Expanding human articular chondrocytes on microcarriers omitting subcultivation steps leads to superior ratios of collagen type II to type I forming cells compared to the expansion in conventional monolayer culture.

Engineering Stem and Stromal Cell Therapies for Musculoskeletal Tissue Repair.

Stem cells and tissue-derived stromal cells stimulate the repair of degenerated and injured tissues, motivating a growing number of cell-based interventions in the musculoskeletal field. Recent investigations have indicated that these cells are critical for their trophic and immunomodulatory role in controlling endogenous cells. This Review presents recent clinical advances where stem cells and stromal cells have been used to stimulate musculoskeletal tissue repair, including delivery strategies to improve cell viability and retention. Emerging bioengineering strategies are highlighted, particularly toward the development of biomaterials for capturing aspects of the native tissue environment, altering the healing niche, and recruiting endogenous cells.

Study of Telomere Length in Preimplanted Cultured Chondrocytes.

In the process of cell division, the extremes of the eukaryotic chromosomes are progressively shortening, and this phenomenon is related to cell degeneration and senescence. The treatment of cartilage lesions with autologous chondrocytes implies that cells proliferate in an artificial environment. We have studied the viability of cultured chondrocytes after measurement of their telomere length before implantation. Articular cartilage biopsies (B1, B2, and B3) were obtained from 3 patients (2 males and 1 female) with knee cartilage defects, who were going to be treated with chondrocyte implantation. Chondrocytes were cultured in DMEM with autologous serum. After the third passage, an aliquot of 1 million cells was removed to estimate the telomere length and the remaining cells were implanted. Telomere length was measured by quantitative fluorescent in situ hybridization (Q-FISH). Patients' clinical outcome was determined preoperatively, and 12 and 24 months postimplantation with the International Knee Documentation Committee (IKDC) questionnaire. After chondrocyte implantation, IKDC score doubled at 12 and 24 months with regard to the basal value. After 3 passages, chondrocytes were cultured for a mean of 45.67 days, the mean duplication time being 4.53 days and the mean number of cell divisions being 10.04 during the culture period. The 20th percentile of telomere lengths were 6.84, 6.96, and 7.06 kbp and the median telomere lengths 10.30, 10.47, and 10.73 kbp, respectively. No significant correlation was found between IKDC score and telomere length. Culturing autologous chondrocytes for implantation is not related to cell senescence in terms of telomere length.

Pentosan Polysulfate Sodium Restores the Phenotype of Dedifferentiated Monolayer Canine Articular Chondrocytes Cultured in Alginate Beads

Autologous chondrocyte transplantation is a promising option for the repair of isolated osteoarthritic cartilage lesions that requires isolation and expansion of chondrocytes from a small cartilage biopsy prior to implantation. However, when cultured in vitro, chondrocytes lose their stable phenotype and dedifferentiate to fibroblastic-like cells. The study investigated the potential of pentosan polysulfate (PPS) sodium to restore the phenotype of dedifferentiated monolayer articular chondrocytes. Canine articular chondrocytes isolated from four cartilage samples were culture expanded to establish primary culture. First passage chondrocytes were cultured as alginate beads for 18 days under normoxia in PPS concentrations of 0, 1, 5, 15 and 40 μg/mL in 20% DMEM. Effect of PPS on type I, II and X collagen, aggrecan and Runx2 gene expression were evaluated by real-time PCR. Runx2, HIF-1α and HIF-2α protein expression were evaluated by Western blot and proteoglycan deposition was determined by Alcian blue stain. Dedifferentiated chondrocytes fully retained their phenotype as evidenced by increased synthesis of cartilage-specific genes, type II collagen and aggrecan mRNA with complete suppression of type I and X collagen at PPS concentrations of 15 and 40 μg/mL. Compared to the control, type II collagen and aggrecan mRNA were significantly upregulated (P<0.05) at 5, 15 and 40 μg/mL and 5 and 15 μg/mL PPS, respectively. PPS significantly enhanced proteoglycan with peak deposition at 5 μg/mL compared to control. HIF-1α and HIF-2α proteins were detectable at protein level for the first time under normoxia condition in alginate culture. The study demonstrates for the first time the restoration of dedifferentiated canine articular chondrocytes phenotype by combining alginate encapsulation with culture in PPS without the addition of known chondrocytic growth factors. The study confirms PPS as novel chondroinductive factor with potential to offer a solution to the major challenges that exist in cartilage tissue engineering.

Chondrocyte-based intraoperative processing strategies for the biological augmentation of a polyurethane meniscus replacement

ABSTRACTPurpose/aim of study: Menisectomies account for over 1.5 million surgical interventions in Europe annually, and there is a growing interest in regenerative strategies to improve outcomes in meniscal replacement. The overall objective of this study was to evaluate the role of intraoperatively applied fresh chondrocyte (FC) isolates compared to minced cartilage (MC) fragments, used without cell isolation, to improve bioactivity and tissue integration when combined with a polyurethane replacement.Materials and methods: First, to optimize the intraoperative cell isolation protocol, caprine articular cartilage biopsies were digested with 750 U/ml or 3000 U/ml collagenase type II (ratio of 10 ml per g of tissue) for 30 min, 1 h or 12 h with constant agitation and compared to culture-expanded chondrocytes in terms of matrix deposition when cultured on polyurethane scaffolds. Finally, FCs and MC-augmented polyurethane scaffolds were evaluated in a caprine meniscal explant model to assess the potential enhancements on tissue integration strength.Results: Adequate numbers of FCs were harvested using a 30 min chondrocyte isolation protocol and were found to demonstrate improved matrix deposition compared to standard culture-expanded cells in vitro. Upon evaluation in a meniscus explant defect model, both FCs and MC showed improved matrix deposition at the tissue-scaffold interface and enhanced push-out strength, fourfold and 2.5-fold, respectively, compared with the acellular implant.Conclusions: Herein, we have demonstrated a novel approach that could be applied intraoperatively, using FCs or MC for improved tissue integration with a polyurethane meniscal replacement.

Culture Medium Supplements Derived from Human Platelet and Plasma: Cell Commitment and Proliferation Support.

Present cell culture medium supplements, in most cases based on animal sera, are not fully satisfactory especially for the in vitro expansion of cells intended for human cell therapy. This paper refers to (i) an heparin-free human platelet lysate (PL) devoid of serum or plasma components (v-PL) and (ii) an heparin-free human serum derived from plasma devoid of PL components (Pl-s) and to their use as single components or in combination in primary or cell line cultures. Human mesenchymal stem cells (MSC) primary cultures were obtained from adipose tissue, bone marrow, and umbilical cord. Human chondrocytes were obtained from articular cartilage biopsies. In general, MSC expanded in the presence of Pl-s alone showed a low or no proliferation in comparison to cells grown with the combination of Pl-s and v-PL. Confluent, growth-arrested cells, either human MSC or human articular chondrocytes, treated with v-PL resumed proliferation, whereas control cultures, not supplemented with v-PL, remained quiescent and did not proliferate. Interestingly, signal transduction pathways distinctive of proliferation were activated also in cells treated with v-PL in the absence of serum, when cell proliferation did not occur, indicating that v-PL could induce the cell re-entry in the cell cycle (cell commitment), but the presence of serum proteins was an absolute requirement for cell proliferation to happen. Indeed, Pl-s alone supported cell growth in constitutively activated cell lines (U-937, HeLa, HaCaT, and V-79) regardless of the co-presence of v-PL. Plasma- and plasma-derived serum were equally able to sustain cell proliferation although, for cells cultured in adhesion, the Pl-s was more efficient than the plasma from which it was derived. In conclusion, the cells expanded in the presence of the new additives maintained their differentiation potential and did not show alterations in their karyotype.

Lubricin binds cartilage proteins, cartilage oligomeric matrix protein, fibronectin and collagen II at the cartilage surface

Lubricin, a heavily O-glycosylated protein, is essential for boundary lubrication of articular cartilage. Strong surface adherence of lubricin is required given the extreme force it must withstand. Disulfide bound complexes of lubricin and cartilage oligomeric matrix protein (COMP) have recently been identified in arthritic synovial fluid suggesting they may be lost from the cartilage surface in osteoarthritis and inflammatory arthritis. This investigation was undertaken to localise COMP-lubricin complexes within cartilage and investigate if other cartilage proteins are involved in anchoring lubricin to the joint. Immunohistochemical analysis of human cartilage biopsies showed lubricin and COMP co-localise to the cartilage surface. COMP knockout mice, however, presented with a lubricin layer on the articular cartilage leading to the further investigation of additional lubricin binding mechanisms. Proximity ligation assays (PLA) on human cartilage biopsies was used to localise additional lubricin binding partners and demonstrated that lubricin bound COMP, but also fibronectin and collagen II on the cartilage surface. Fibronectin and collagen II binding to lubricin was confirmed and characterised by solid phase binding assays with recombinant lubricin fragments. Overall, COMP, fibronectin and collagen II bind lubricin, exposed on the articular cartilage surface suggesting they may be involved in maintaining essential boundary lubrication.

Two-stage late reconstruction with a fresh large osteochondral shell allograft transplantation (FLOCSAT) for a large ostechondral defect in a non-union after a lateral tibia plateau fracture 2-year follow up

This is the description of a 58-year-old female patient presenting 8 months after a horse riding accident with significant pain and inability to walk independently. Imaging revealed a large osseous defect of the lateral tibia plateau which was not united posteriorly. The patient refused knee replacement and we developed a patient specific two-step procedure for her. Step 1: Filling of the defect with a large cortico-cancellous autograft from the posterior iliac crest; step 2: Transplantation of a fresh large osteochondral shell allograft (FLOCSAT). The postoperative protocol included continuous passive motion (CPM), partial weight bearing for three months, and physiotherapy. Based on the concept of immuno-privileged cartilage tissue, the patient did not get any immuno-suppressive therapy.Pain-, activity of daily living, Lysholm and Tegner scores were evaluated before defect filling surgery with autograft, before allograft transplantation, and at 12 and 24 months after allograft transplantation. There were no complications.Radiographic analyses with plain films and CT scans revealed solid osseous integration within 3 month. The patient regained excellent functionality in both, activities of daily living and sports (back to horse riding, trampolin jumping). Knee arthroscopy after 1year showed excellent condition of the lateral meniscus and the cartilage of the lateral tibia plateau. Chimerism/DNA analysis of a cartilage biopsy showed, that at 1year 32% of the donor cells have been already replaced by the patient’s own cells.To our knowledge, this is the first case of a patient who sustained such a large defect during a tibia plateau fracture, and got successfully treated with a fresh large osteochondral shell allograft transplantation in a two-step procedure.

Safety of three different product doses in autologous chondrocyte implantation: results of a prospective, randomised, controlled trial

BackgroundThis study was conducted to assess the efficacy and safety of the three dose levels of the three-dimensional autologous chondrocyte implantation product chondrosphere® in the treatment of cartilage defects (4–10 cm2) of knee joints. We hereby report the safety results for a 36-month post-treatment observation period.MethodsThis was a prospective phase II trial with a clinical intervention comprising biopsy for culturing spheroids and their subsequent administration (level of evidence: I). Patients’ knee defects were investigated by arthroscopy, and a cartilage biopsy was taken for culturing. Patients were randomised, on a single-blind basis, to treatment at the dose levels 3–7 (low), 10–30 (medium) or 40–70 (high) spheroids per square centimetre. Assessment (adverse events, vital signs, electrocardiography, physical examination, concomitant medication and laboratory values) took place 1.5, 3, 6, 12, 24 and 36 months after chondrocyte implantation.ResultsSeventy-five patients were included and 73 treated. The incidence of adverse events, of patients with adverse events and of patients with treatment-related adverse events showed no relevant difference between the treatment groups. There were no fatal adverse events, no adverse events led to premature withdrawal from the trial and none led to permanent sequelae. Two patients experienced serious adverse events considered related to the study treatment: arthralgia 2–3 years after implantation and chondropathy 1 and 2 years after implantation.ConclusionsThe treatment with chondrosphere® was generally well tolerated. No relationship was detected between any safety criteria and the dose level: Differences between the dose groups in the incidence of any adverse events, and in numbers of patients with treatment-related adverse events, were insubstantial.Trial registrationclinicaltrials.gov, NCT01225575.

Measurement of telomere lenght as a marker of senescence in human cultured chondrocytes

Purpose: Autologous chondrocyte implantation is one of the treatments for cartilage repair. It is considered that a telomere shorter than 3 Kbp (kilo base-pairs) is a critically short telomere related to fusion events that lead to genome instability and cell senescence. In this work, the proliferative ability of cultured chondrocytes, in terms of their telomere length, before being implanted to patients with cartilage defects, was studied. Methods: Articular cartilage biopsies (B1, B2 and B3) were obtained from 3 patients (2 males and 1 female) with a similar age (36, 40 and 42 years-old) with cartilage defects in the knee, which were going to be treated with high density autologous chondrocyte (ICC). The chondrocytes were isolated and cultured in DMEM with autologous serum. After the 3rd passage, an aliquot of 1 million cells was removed to estimate the telomere length and the remaining cells were implanted in the patients. In each culture, the doubling time and the estimated number of cell divisions during expansion were calculated. Telomere length measurement was carried-out by quantitative fluorescent in situ hybridization (Q-FISH), studying 10 metaphases from each cell culture. Results: The number of the starting cells obtained was 10000 in B1, 45000 in B2 and 38000 chondrocytes in B3, and after 53, 49 and 35 days of culture, a number of 20, 38 and 26 million cells were obtained, respectively. The doubling time and the number of cell divisions are shown in the table 1. The figure 1 shows a representative Q-FISH from each cartilage biopsy. The median telomere length in each cell culture was 10.3, 10.7 and 10.5 Kbp, respectively (Table 2), being these values statically higher than 3 Kbp (p&lt0.001) in the 3 cases. Conclusions: Telomeres of the chondrocyte cultured for future implantation are larger enough to think that these cells are not senescent.Table 1Doubling time and number of cell divisions during the culturesCartilage biopsyAge (years)GenderTime in culture (days)Dubling time (days)Estimated number of eel divisionsB140Female534.8310.97B236Male495.049.72B342Male353.729.42 Open table in a new tab Table 2Median and 20 th percentile of telomere length of the cartillage biopsiesCartilage biopsyMedian lenght (bp)20 th Percentile lenght (bp)Coeficient of variationB110.2706.8403.94B210.7337.0955.72B310.4677.0593.54 Open table in a new tab

* Human Amniotic Mesenchymal Stromal Cells as Favorable Source for Cartilage Repair.

Localized trauma-derived breakdown of the hyaline articular cartilage may progress toward osteoarthritis, a degenerative condition characterized by total loss of articular cartilage and joint function. Tissue engineering technologies encompass several promising approaches with high therapeutic potential for the treatment of these focal defects. However, most of the research in tissue engineering is focused on potential materials and structural cues, while little attention is directed to the most appropriate source of cells endowing these materials. In this study, using human amniotic membrane (HAM) as scaffold, we defined a novel static in vitro model for cartilage repair. In combination with HAM, four different cell types, human chondrocytes, human bone marrow-derived mesenchymal stromal cells (hBMSCs), human amniotic epithelial cells, and human amniotic mesenchymal stromal cells (hAMSCs) were assessed determining their therapeutic potential. A chondral lesion was drilled in human cartilage biopsies simulating a focal defect. A pellet of different cell types was implanted inside the lesion and covered with HAM. The biopsies were maintained for 8 weeks in culture. Chondrogenic differentiation in the defect was analyzed by histology and immunohistochemistry. HAM scaffold showed good integration and adhesion to the native cartilage in all groups. Although all cell types showed the capacity of filling the focal defect, hBMSCs and hAMSCs demonstrated higher levels of new matrix synthesis. However, only the hAMSCs-containing group presented a significant cytoplasmic content of type II collagen when compared with chondrocytes. More collagen type I was identified in the new synthesized tissue of hBMSCs. In accordance, hBMSCs and hAMSCs showed better International Cartilage Research Society scoring although without statistical significance. HAM is a useful material for articular cartilage repair in vitro when used as scaffold. In combination with hAMSCs, HAM showed better potential for cartilage repair with similar reparation capacity than chondrocytes.

Morphological Study: Ultrastructural Aspects of Articular Cartilage and Subchondral Bone in Patients Affected by Post-Traumatic Shoulder Instability.

Post-traumatic shoulder instability is a frequent condition in active population, representing one of most disabling pathologies, due to altered balance involving joints. No data are so far available on early ultrastructural osteo-chondral damages, associated with the onset of invalidating pathologies, like osteoarthritis-OA. Biopsies of glenoid articular cartilage and sub-chondral bone were taken from 10 adult patients underwent arthroscopic stabilization. Observations were performed under Transmission Electron Microscopy-TEM in tangential, arcuate and radial layers of the articular cartilage and in the sub-chondral bone. In tangential and arcuate layers chondrocytes display normal and very well preserved ultrastructure, probably due to the synovial liquid supply; otherwise, throughout the radial layer (un-calcified and calcified) chondrocytes show various degrees of degeneration; occasionally, in the radial layer evidences of apoptosis/autophagy were also observed. Concerning sub-chondral bone, osteocytes next to the calcified cartilage also show signs of degeneration, while osteocytes farther from the osteo-chondral border display normal ultrastructure, probably due to the bone vascular supply. The ultrastructural features of the osteo-chondral complex are not age-dependent. This study represents the first complete ultrastructural investigation of the articular osteo-chondral complex in shoulder instability, evaluating the state of preservation/viability of both chondrocytes and osteocytes throughout the successive layers of articular cartilage and sub-chondral bone. Preliminary observations here collected represent the morphological basis for further deepening of pathogenesis related to shoulder instability, enhancing the relationship between cell shape and microenvironment; in particular, they could be useful in understanding if the early surgical treatment in shoulder instability could avoid the onset of OA. Anat Rec, 300:1208-1218, 2017. © 2016 Wiley Periodicals, Inc.

Intra-articular Xenotransplantation of Adipose-Derived Stromal Cells to Treat Osteoarthritis in a Goat Model.

Adipose-derived stromal cells (ASCs) have been investigated as a cell source for tissue regeneration. The purpose of this study was first to confirm if medial meniscectomy induces osteoarthritis (OA) in goats within a relative short period of time, and more importantly, to investigate if systemic treatment with immunosuppressive drugs is necessary in intra-articular ASC xenotransplantation for successful regeneration of articular cartilage and prevention of joint inflammation. Eight Korean native black goats 1-2 years of age underwent medial meniscectomy. To evaluate the gross and histological appearance of articular cartilage, knee joints were re-exposed by a medial parapatellar incision at 8 weeks. After macroscopic scoring of gross appearance, cartilage biopsy specimens 6 mm in diameter were obtained from the femoral condyle in four goats. The goats were injected with single intra-articular dose of 7×106 human ASCs (hASCs) 7 days after the second arthrotomy. Four animals were treated with daily injections of cyclosporin A 10 mg/kg for 7 days, followed by a reduced dose of 5 mg/kg for another 7 days, while other 4 animals did not receive immunosuppressive therapy. All animals were sacrificed for analysis 8 weeks after injection of hASCs. OA was successfully induced 8 weeks after medial meniscectomy. Eight weeks after injection of hASCs, various signs of articular cartilage regeneration were observed. There were no significant macroscopic and histological differences between goats treated with cyclosporine and untreated goats. Interleukin-1ß and tumor necrosis factor-α level from synovial fluid did not differ between cyclosporine-treated and untreated goats. The results indicate that immunosuppressive therapy did not influence the result of ASC xenotransplantation to treat OA.

Impact of expansion and redifferentiation under hypothermia on chondrogenic capacity of cultured human septal chondrocytes

A critical limitation in the cultivation of cartilage for tissue engineering is the dedifferentiation in chondrocytes, mainly during in vitro amplification. Despite many previous studies investigating the influence of various conditions, no data exist concerning the effects of hypothermia. Our aim has been to influence chondrocyte dedifferentiation in vitro by hypothermic conditions. Chondrocytes were isolated from cartilage biopsies and seeded in monolayer and in three-dimensional pellet-cultures. Each cell culture was either performed at 32.2°C or 37°C during amplification. Additionally, the influence of the redifferentiation of chondrocytes in three-dimensional cell culture was examined at 32.2°C and 37°C after amplification at 32.2°C or 37°C. An 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay was used to measure cell proliferation in monolayer, whereas the polymerase chain reaction and immunohistochemical and histological staining were used in three-dimensional pellet-cultures. Real-time polymerase chain reaction was employed to measure the relative expression of the target genes collagen II, collagen I, aggrecan and versican. Ratios were estimated between collagen II/collagen I and aggrecan/versican to evaluate differentiation. A higher value of these ratios indicated an advantageous status of differentiation. In monolayer, hypothermia at 32.2°C slowed down the proliferation rate of chondrocytes significantly, being up to two times lower at 32.2°C compared with culture at 37°C. Simultaneously, hypothermia in monolayer decelerated dedifferentiation. The ratio of aggrecan/versican was significantly higher at 32.2°C compared with that at 37°C. In three-dimensional pellet-culture, the chondrocytes redifferentiated at 32.2°C and at 37°C, and this process is more distinct at 37°C than at 32.2°C. Similar results were obtained for the ratios of collagen II/collagen I and aggrecan/versican and were supported by immunochemical and histological staining. Thus, hypothermic conditions for chondrocytes are mainly advantageous in monolayer culture. In three-dimensional pellet-culture, redifferentiation predominates at 37°C compared with at 32.2°C. In particular, the results from the monolayer cultures show potential in the avoidance of dedifferentiation.

Human Cartilage Engineering in an In Vitro Repair Model Using Collagen Scaffolds and Mesenchymal Stromal Cells.

The purpose of this study was to investigate cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (hBMSCs) with different collagen (Col) scaffolds.Lesions were made in human cartilage biopsies. Injured samples were pre-treated with interleukin 1β (IL1β) for 24 h; also, samples were not pre-treated. hBMSCs were seeded on different types of collagen scaffolds. The resulting constructs were placed into the lesions, and the biopsies were cultured for 2 months in chondrogenic medium.Using the modified ICRSII scale, neotissues from the different scaffolds showed ICRS II overall assessment scores ranging from 50% (fibrocartilage) to 100% (hyaline cartilage), except for the Col I +Col II +HS constructs (fibrocartilage/hyaline cartilage, 73%). Data showed that hBMSCs cultured only on Col I +Col II +HS scaffolds displayed a chondrocyte-like morphology and cartilage-like matrix close to native cartilage. Furthermore, IL1β pre-treated biopsies decreased capacity for repair by hBMSCs and decreased levels of chondrogenic phenotype of human cartilage lesions.