Infrapatellar Fat Pad Modulates Osteoarthritis-Associated Cytokine and MMP Expression in Human Articular Chondrocytes.

Osteoarthritis (OA) most frequently affects the knee joint and is associated with an elevated expression of cytokines and extracellular cartilage matrix (ECM), degrading enzymes such as matrix metalloproteinases (MMPs). Differences in gene expression of the intra-articularly located infrapatellar fat pad (IPFP) and other fatty tissue suggest its autonomous function, yet its role in OA pathogenesis remains unknown. Human IPFPs and articular cartilage were collected from OA patients undergoing total knee arthroplasty, and biopsies from the IPFP of healthy patients harvested during knee arthroscopy served as controls (CO). Isolated chondrocytes were co-cultured with either osteoarthritic (OA) or CO-IPFPs in a transwell system. Chondrocyte expression of MMP1, -3, -13, type 1 and 2 collagens, interleukin IL1ß, IL6, IL10, and tumor necrosis factor TNFα was analyzed by RTD-PCR at day 0 and day 2, and TNFα secretion was analyzed by ELISA. The cytokine release in IPFPs was assessed by an array. Results: Both IPFPs (CO, OA) significantly reduced the expression of type 2 collagen and TNFα in chondrocytes. On the other hand, only CO-IPFP suppressed the expression of type 1 collagen and significantly induced the MMP13 expression. On the contrary, IL1ß and IL6 were significantly induced when exposed to OA-IPFP. Conclusions: The partial loss of the suppressive effect on type 1 collagen gene expression found for OA-IPFP shows the pathological remodeling and dedifferentiation potential of the OA-IPFP on the chondrocytes. However, the significant suppression of TNFα implies that the OA- and CO-IPFP could also exhibit a protective role in the knee joint, preventing the progress of inflammation.

Comparison of accuracy of two uncalibrated pulse contour cardiac output monitors in off-pump coronary artery bypass surgery patients using pulmonary artery catheter-thermodilution as a reference.

BACKGROUND: Cardiac output (CO) is a key measure of adequacy of organ and tissue perfusion, especially in critically ill or complex surgical patients. CO monitoring technology continues to evolve. Recently developed CO monitors rely on unique algorithms based on pulse contour analysis of an arterial blood pressure (ABP) waveform. The objective of this investigation was to compare the accuracy of two monitors using different methods of pulse contour analysis - the Retia Argos device and the Edwards Vigileo-FloTrac device - with pulmonary artery catheter (PAC)-thermodilution as a reference. METHODS: Fifty-eight patients undergoing off-pump coronary artery bypass surgery formed the study cohort. A total of 572 triplets of CO measurements from each device - Argos, Vigileo-FloTrac (third generation), and thermodilution - were available before and after interventions (e.g., vasopressors, fluids, and inotropes). Bland-Altman analysis accounting for repeated measurements per subject and concordance analysis were applied to assess the accuracy of the CO values and intervention-induced CO changes of each pulse contour device against thermodilution. Cluster bootstrapping was employed to statistically compare the root-mean-squared-errors (RMSE = √(µ2 + σ2), where µ and σ are the Bland-Altman bias and precision errors) and concordance rates of the two devices. RESULTS: The RMSE (mean (95% confidence intervals)) for CO values was 1.16 (1.00-1.32) L/min for the Argos device and 1.54 (1.33-1.77) L/min for the Vigileo-FloTrac device; the concordance rate for intervention-induced CO changes was 87 (82-92)% for the Argos device and 72 (65-78)% for the Vigileo-FloTrac device; and the RMSE for the CO changes was 17 (15-19)% for the Argos device and 21 (19-23)% for the Vigileo-FloTrac device (p < 0.0167 for all comparisons). CONCLUSIONS: In comparison with CO measured by the PAC, the Argos device proved to be more accurate than the Vigileo-FloTrac device in CO trending and absolute CO measurement in patients undergoing off-pump coronary artery bypass surgery.

Complement Regulation in Human Tenocytes under the Influence of Anaphylatoxin C5a.

A central part of the complement system, the anaphylatoxin C5a was investigated in this study to learn its effects on tenocytes in respect to understanding the potential expression of other crucial complement factors and pro-inflammatory mediators involved in tendinopathy. Human hamstring tendon-derived tenocytes were treated with recombinant C5a protein in concentrations of 25 ng/mL and 100 ng/mL for 0.5 h (early phase), 4 h (intermediate phase), and 24 h (late phase). Tenocytes survival was assessed after 24 h stimulation by live-dead assay. The gene expression of complement-related factors C5aR, the complement regulatory proteins (CRPs) CD46, CD55, CD59, and of the pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-6 was monitored using qPCR. Tenocytes were immunolabeled for C5aR and CD55 proteins. TNFα production was monitored by ELISA. Tenocyte survival was not impaired through C5a stimulation. Interestingly, the gene expression of C5aR and that of the CRPs CD46 and CD59 was significantly reduced in the intermediate and late phase, and that of TNFα only in an early phase, compared to the control group. ELISA analysis indicated a concomitant not significant trend of impaired TNFα protein synthesis at 4 h. However, there was also an early significant induction of CD55 and CD59 mediated by 25 ng/mL anaphylatoxin C5a. Hence, exposure of tenocytes to C5a obviously evokes a time and concentration-dependent response in their expression of complement and pro-inflammatory factors. C5a, released in damaged tendons, might directly contribute to tenocyte activation and thereby be involved in tendon healing and tendinopathy.

Complement regulation in tenocytes under the influence of leukocytes in an indirect co-culture model.

INTRODUCTION: The present in vitro study was undertaken to learn about the effects of leukocytes on tenocytes in respect to complement regulation simulating an inflammatory scenario of the traumatized tissue. METHODS: Human hamstring tendon-derived tenocyte monolayers were co-cultured indirectly with human leukocytes (either Peripheral Blood Mononuclear Cells [PBMCs] or neutrophils) using a transwell system with/without (+ /wo) 10 ng/ml tumor necrosis factor α (TNFα) for 4 and 24 h. Tenocyte and leukocyte cell survival was assessed by live-dead assay. Tenocyte gene expression of TNFα, the anaphylatoxin receptor C5aR and the cytoprotective complement regulatory proteins (CRP) CD46, CD55 and CD59 was monitored using qPCR. TNFα was detected in the culture supernatants using ELISA. RESULTS: C5aR gene expression was significantly induced by TNFα after 4 h, but impaired in the presence of leukocytes + TNFα after 24 h. At 4 h, PBMCs activated by TNFα induced the CRP CD46 gene expression. However, CD55 was significantly suppressed after 24 h by neutrophils + /woTNFα. Leukocytes activated by TNFα decreased also significantly the gene expression of the more downstream acting CRP CD59 after 4 h. TNFα gene expression and ELISA analysis revealed an amplified TNFα expression/release in tenocyte co-cultures with PBMC + /woTNFα, probably contributing to complement regulation. CONCLUSION: TNFα might represent a crucial soluble mediator exerting diverse time-dependent effects on tenocyte complement regulation.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

SV40 Transfected Human Anterior Cruciate Ligament Derived Ligamentocytes-Suitable as a Human in Vitro Model for Ligament Reconstruction?

Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.

ECMO used successfully in a near fatal case of opioid-induced acute respiratory distress syndrome.

Acute Respiratory Distress Syndrome (ARDS) was first recognized during the 1960s. It is a distinct type of hypoxemic respiratory failure characterized by acute abnormality of both lungs. Extracorporeal membrane oxygenation (ECMO) is being increasingly used for patients with severe ARDS refractory to otherwise conventional management. A 29year old male arrived with Emergency Medical Services (EMS) status post presumed heroin overdose. He was administered Naloxone 2mg intravenously prior to arrival in the emergency department. The patient arrived in severe respiratory distress with a pulse oximetry level of 50% and was immediately intubated. The patient's pulse oximetry level remained in the seventies despite intubation and aggressive ventilator management. The Intensive Care Unit team in conjunction with cardiothoracic surgery initiated venovenous ECMO therapy in the emergency department itself. The patient was transferred to a tertiary center for venoarterial ECMO that was continued for 6 more days. After an extensive hospitalization, the patient was ultimately transferred to an acute medical rehabilitation center. With the current opioid crisis, emergency physicians and providers need to be aware that opioids can induce severe ARDS refractory to mechanical ventilation. ECMO as a treatment option can be used safely and successfully as described in this unique patient case report.

Coexisting Cardiac and Hematologic Disorders.

Patients with concomitant cardiac and hematologic disorders presenting for noncardiac surgery are challenging. Anemic patients with cardiac disease should be approached in a methodical fashion. Transfusion triggers and target should be based on underlying symptomatology. The approach to anticoagulation management in patients with artificial heart valves, cardiac devices, or severe heart failure in the operative setting must encompass a complete understanding of the rationale of a patient's therapy as well as calculate the risk of changing this regimen. This article focuses common disorders and discusses strategies to optimize care in patients with coexisting cardiac and hematologic disease.

Establishment of a Cytocompatible Cell-Free Intervertebral Disc Matrix for Chondrogenesis with Human Bone Marrow-Derived Mesenchymal Stromal Cells.

Tissue-engineered intervertebral discs (IVDs) utilizing decellularized extracellular matrix (ECM) could be an option for the reconstruction of impaired IVDs due to degeneration or injury. The objective of this study was to prepare a cell-free decellularized human IVD scaffold and to compare neotissue formation in response to recellularization with human IVD cells (hIVDCs) or human bone marrow-derived (hBM) mesenchymal stromal cells (MSCs). IVDs were decellularized via freeze-thaw cycles, detergents and trypsin. Histological staining was performed to monitor cell removal and glycosaminoglycan (GAG) removal. The decellularized IVD was preconditioned using bovine serum albumin and fetal bovine serum before its cytocompatibility for dynamically cultured hBM-MSCs (chondrogenically induced or not) and hIVDCs was compared after 14 days. In addition, DNA, total collagen and GAG contents were assessed. The decellularization protocol achieved maximal cell removal, with only few remaining cell nuclei compared with native tissue, and low toxicity. The DNA content was significantly higher in scaffolds seeded with hIVDCs compared with native IVDs, cell-free and hBM-MSC-seeded scaffolds (p < 0.01). The GAG content in the native tissue was significantly higher compared to the others groups except for the scaffolds reseeded with chondrogenically induced hBM-MSCs (p < 0.05). In addition, there was a significantly increased total collagen content in the chondrogenically induced hBM-MSCs group (p < 0.01) compared with the native IVDs, cell-free and hIVDC-seeded scaffolds (p < 0.01); both recolonizing cell types were more evenly distributed on the scaffold surface, but only few cells penetrated the scaffold. The resulting decellularized ECM was cytocompatible and allowed hBM-MSCs/hIVDCs survival and ECM production.

Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate.

To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1-2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~93-95% with a mean pore sizes of 237±48 µm (Alg) and 197±61 µm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell-cell contacts.

Effect of nasal sprays on an in vitro survival and morphology of nasoseptal cartilage.

Nasal sprays were introduced several years ago to support the treatment of allergic rhinitis. These sprays may come in direct contact with directly exposed nasoseptal cartilage (e.g. is case of nasoseptal perforation). To date, no studies investigated the effects of nasal sprays on cartilage tissues and cells. Therefore, our aim was to analyze the influence of two different nasal spray types (thixotropic and liposomal) on the vitality of nasoseptal chondrocytes. Human chondrocytes were isolated from surgically dissected tissues. Alternatively, nasal septa (porcine and human) tissue explants were used. The cell or explant cultures were treated with nasal sprays for 4-24 h. As a read-out, cell vitality and gene and protein expression profiles of type I and II collagen, SOX 9 and matrix metalloproteinase MMP-1 were compared to the untreated controls by means of real-time RT-PCR and immunostaining. Using the liposomal, but not thixotropic nasal spray in an explant or chondrocyte in vitro culture led to increased cell death, as compared to the untreated controls. A trend towards suppression of type II collagen and SOX 9 on protein level was found in cultures exposed to liposomal nasal spray, as compared to the controls. The thixotropic nasal spray has not affected the nasoseptal chondrocytes. Further studies with the use of viable nasoseptal cartilage explants and particularly using an in vivo animal model of exposed nasoseptal cartilage are necessary to clear the effect of liposomal spray on chondrocytes.

Tenocyte activation and regulation of complement factors in response to in vitro cell injury.

Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)α, interleukin (IL)-1ß, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was down-regulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNFα gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses.

There's no place like home: boarding surgical ICU patients in other ICUs and the effect of distances from the home unit.

BACKGROUND: Intensive care units (ICUs) function frequently at capacity, requiring incoming critically ill patients to be placed in alternate geographically distinct ICUs. In some medical ICU populations, "boarding" in an overflow ICU has been associated with increased mortality. We hypothesized that surgical ICU patients experience more complications when boarding in an overflow ICU and that the frequency of these complications are greatest in boarders farthest from the home unit (HU). METHODS: A 5-year (June 2005 to June 2010) retrospective review of a prospectively maintained ICU database was performed, and demographics, severity of illness, length of stay, and incidence of ICU complications were extracted. Distances between boarding patients' rooms and the HU were measured. Complications occurring in patients located in the same floor (BUSF) and different floor (BUDF) boarding units were compared and stratified by distance from HU to the patient room. Logistic regression was used to develop control for known confounders. RESULTS: A total of 7,793 patients were admitted to the HU and 833 to a boarding unit (BUSF, n = 712; BUDF, n = 121). Boarders were younger, had a lower length of stay, and Acute Physiology and Chronic Health Evaluation II and were more often trauma/emergency surgery patients. Compared with in-HU patients, the incidence of aspiration pneumonia (2.2% vs. 3.6%, p < 0.01) was greater in BUSF patients and highest in those farthest from the HU (odds ratio [OR], 2.39; p = 0.01). Delirium occurred less often in HU than in BUDF patients (3.3% vs. 8.3 %, p < 0.01), and both delirium (OR, 6.09, p < 0.01) and ventilator-associated pneumonia (OR, 4.49, p < 0.05) were more frequent in patients farther from the HU. CONCLUSION: Certain ICU complications occur more frequently in boarding patients particularly if they are located on a different floor or far from the HU. When surgical ICU bed availability forces overflow admissions to non-home ICUs, greater interdisciplinary awareness, education, and training may be needed to ensure equivalent care and outcomes. LEVEL OF EVIDENCE: Epidemiologic study, level III. Therapeutic study, level IV.

The year in cardiothoracic and vascular anesthesia: selected highlights from 2013.

This article reviewed selected research highlights of 2013 that pertain to the specialty of cardiothoracic and vascular anesthesia. The first major theme is the commemoration of the sixtieth anniversary of the first successful cardiac surgical procedure with cardiopulmonary bypass conducted by Dr Gibbon. This major milestone revolutionized the practice of cardiovascular surgery and invigorated a paradigm of mechanical platforms for contemporary perioperative cardiovascular practice. Dr Kolff was also a leading contributor in this area because of his important contributions to the refinement of cardiopulmonary bypass and mechanical ventricular assistance. The second major theme is the diffusion of echocardiography throughout perioperative practice. There are now guidelines and training pathways to guide its generalization into everyday practice. The third major theme is the paradigm shift in perioperative fluid management. Recent large randomized trials suggest that fluids are drugs that require a precise prescription with respect to type, dose, and duration. The final theme is patient safety in the cardiac perioperative environment. A recent expert scientific statement has focused attention on this issue because most perioperative errors are preventable. It is likely that clinical research in this area will blossom because this is a major opportunity for improvement in our specialty. The patient care processes identified in these research highlights will further improve perioperative outcomes for our patients.

Intravenous GLP-1 (7-36) amide for prevention of hyperglycemia during cardiac surgery: a randomized, double-blind, placebo-controlled study.

OBJECTIVE: The authors sought to evaluate the efficacy of an intravenous glucagon-like peptide-1 (GLP-1) infusion, compared with placebo, to mitigate intraoperative hyperglycemia. DESIGN: Prospective, double-blinded, randomized, placebo-controlled. SETTING: University hospital. PARTICIPANTS: Diabetic (non-insulin dependent) and non-diabetic patients undergoing elective cardiac surgery with cardiopulmonary bypass. INTERVENTIONS: Patients were randomized in a 1:1 fashion to GLP-1 (7-36) amide infusion (1.5 pmol/kg/min) or placebo. Insulin was administered intraoperatively to both groups per a standardized protocol. MEASUREMENTS AND MAIN RESULTS: A total of 77 patients were included for analysis (GLP-1, n = 37; placebo, n = 40). Mean blood glucose during cardiopulmonary bypass was 127.5 mg/dL and 142.5 mg/dL (p = 0.002) in the GLP-1 and placebo groups, respectively. Mean blood glucose values during the entire intraoperative course were 12.2 mg/dL lower for subjects given GLP-1 (95% CI 2.3, 22, p = 0.015), independent of time. During the period of cardiopulmonary bypass, mean blood glucose values in subjects given GLP-1 were 14.1 mg/dL lower than those who received placebo (95% CI 3.5, 24.8, p = 0.009), independent of time. The incidence of hypoglycemia did not differ significantly between the 2 groups. CONCLUSIONS: Administration of intravenous GLP-1 (7-36) amide to patients undergoing cardiac surgery significantly reduced their plasma glucose levels intraoperatively and may represent a novel therapeutic strategy to prevent perioperative hyperglycemia.

Selectivity in bone targeting with multivalent dendritic polyanion dye conjugates.

Targeting bone with anionic macromolecules is a potent approach for the development of novel diagnostics and therapeutics for bone related diseases. A highly efficient modular synthesis of dendritic polyglycerol (dPG) polyanion dye conjugates, namely, sulfates, sulfonates, carboxylates, phosphates, phosphonates, and bisphosphonates via click chemistry is presented. By investigating the microarchitecture of stained bone sections with confocal laser scanning microscopy, the bisphosphonate, phosphonate, and phosphate functionalized polymers are identified as strongly penetrating compounds, whereas sulfates, sulfonates, and carboxylates reveal a weaker binding to hydroxyapatite (HA) but a more pronounced affinity toward collagen. In a quantitative HA binding assay, the affinity of the dPG sulfonate, sulfate, and carboxylate toward collagen and the exceptional high HA affinity of the phosphorous containing polyelectrolytes are validated. This shows the potential of dendritic polyphosphates and phosphonates as alternatives to the commonly employed bisphosphonate modification. In cytotoxicity studies with murine fibroblasts, the conjugates have no significant effect on the cell viability at 10(-5) m. All polyanions are taken up into the cells within 24 h. The presented synthetic approach allows versatile extensions for preparing conjugates for selective bone imaging applications, tissue engineering, and drug delivery.

Effect of polystyrene and polyether imide cell culture inserts with different roughness on chondrocyte metabolic activity and gene expression profiles of aggrecan and collagen.

In vitro cultured autologous chondrocytes can be used for implantation to support cartilage repair. For this purpose, a very small number of autologous cells harvested from a biopsy have to be expanded in monolayer culture. Commercially available polymer surfaces lead to chondrocyte dedifferentiation. Hence, the demanding need for optimized polymers and surface topologies supporting chondrocytes' differentiated phenotypes in vitro arises. In this study we explored the effect of tailored cell culture plate inserts prepared from polystyrene (PS) and polyether imide (PEI) exhibiting three different roughness levels (R0, RI, RII) on chondrocyte morphology, metabolism and gene expression profile. As a control, commercially available tissue culture plastic (TCP) dishes were included. Primary porcine articular chondrocytes were seeded on tailored PS and PEI inserts with three different roughness levels. The metabolic activity of the chondrocytes was determined after 24 hours using alamar blue assay. Chondrocyte gene expression profiles (aggrecan, type I and type II collagen) were monitored after 48 hours using Real Time Detection (RTD)-PCR. Chondrocytes cultured on PS and PEI surfaces formed cell clusters after 24 and 48 hours, which was not observed on TCP. The metabolic activity of chondrocytes cultured on PS was lower than of chondrocytes cultured on PEI, but also lower than on TCP. Gene expression analyses revealed an elevated expression of cartilage-specific aggrecan and an impaired expression of both collagen types by chondrocytes on PS and PEI compared with TCP. In summary, PEI is a biocompatible biomaterial suitable for chondrocyte culturing, which can be further chemically functionalized for generating specific surface interactions or covalent binding of biomolecules.

Heterotopic and orthotopic autologous chondrocyte implantation using a minipig chondral defect model.

Implantation of non-articular (heterotopic) chondrocyte-based implants might be an alternative approach to articular cartilage repair. This strategy could be helpful in cases in which there are no or too few articular chondrocytes available. Therefore, this study was undertaken to compare joint cartilage defect healing in the minipig model after implantation of heterotopic auricular and orthotopic articular chondrocytes. Poly-glycolic acid (PGA) associated three-dimensional (3D) constructs were prepared culturing autologous minipig-derived articular and auricular chondrocytes for 7 days in a dynamic culture system. Chondrocyte PGA constructs were implanted into 8mm diameter and ∼1.1mm deep chondral defects within the medial and lateral condyles of the minipig knee joints. Empty defects served as controls for assessment of the intrinsic healing response. Defect healing was monitored 6 months post implantation using a macroscopic and microscopic score system and biomechanical analysis. Neo-cartilage formation could be observed in the PGA constructs seeded with articular and auricular chondrocytes in vivo. The defect healing did not significantly differ at the macroscopic and histological level in response to implantation of either autologous articular or auricular chondrocytes seeded constructs compared with the empty defects. Although the differences were not significant, the auricular chondrocytes-based implants led to a slightly inferior repair quality at the macroscopic level, but a histologically superior healing response when compared with the empty defect group. However, biomechanical analysis revealed a higher stiffness in repair tissues produced by auricular chondrocyte implantation compared with the other groups. Deduced from these results, articular chondrocytes represent the preferable cell source for implantation.