D.B. Saris

Patient Profiling in Cartilage Regeneration Prognostic Factors Determining Success of Treatment for Cartilage Defects

Background Cartilage therapy for focal articular lesions has been implemented for more than a decade, and it is becoming increasingly available. What is still lacking, however, is analysis of patient characteristics to help improve outcome or select patients for specific treatment. Purpose To analyze the prognostic value of patient age and defect size, age, and location on clinical outcome 3 years after cartilage therapy. Study Design Cohort study; Level of evidence, 3. Methods Fifty-five patients (age, 35 ± 9 years) were randomly selected from a prospective database. Each had a traumatic knee injury, each was treated for a focal cartilage lesion, and each was assessed with the Knee injury and Osteoarthritis Outcome Score (KOOS) 3 years after surgery. Patient characteristics (ie, patient age and defect size, age, and location) were tested for valid inclusion in the regression model. Multiple linear regression was used to determine which variables influenced clinical improvement. Binary KOOS scores were generated on the basis of age-matched healthy patients and assessed in a logistic regression analysis. Results Normality tests confirmed normal distribution for each variable (P < .05). Defect size did not influence clinical improvement (P > .05). Clinical outcome regarding the treatment of medial defects was better than that of the lateral defects (10.38-25.26 points for the different KOOS subscales; P < .05). The KOOS improvement from baseline was better for patients <30 years compared with patients >30 years (7.31-29.24 points for the different KOOS subscales; P <.05). Patients with defects <24 months were more likely to report the age-matched healthy reference KOOS (odds ratio, 1.8-4.0; P <.05). Conclusion This study illustrates the influence of patient age and defect location and age on clinical outcome 3 years after treatment of a focal cartilage lesion in patients with a traumatic knee injury.

The Effect of Timing of Mechanical Stimulation on Proliferation and Differentiation of Goat Bone Marrow Stem Cells Cultured on Braided PLGA Scaffolds

Bone marrow stromal cells (BMSCs) have been shown to proliferate and produce matrix when seeded onto braided poly(L-lactide/glycolide) acid (PLGA) scaffolds. Mechanical stimulation may be applied to stimulate tissue formation during ligament tissue engineering. This study describes for the first time the effect of constant load on BMSCs seeded onto a braided PLGA scaffold. The seeded scaffolds were subjected to four different loading regimes: Scaffolds were unloaded, loaded during seeding, immediately after seeding, or 2 days after seeding. During the first 5 days, changing the mechanical environment seemed to inhibit proliferation, because cells on scaffolds loaded immediately after seeding or after a 2-day delay, contained fewer cells than on unloaded scaffolds or scaffolds loaded during seeding (p<0.01 for scaffolds loaded after 2 days). During this period, differentiation increased with the period of load applied. After day 5, differences in cell content and collagen production leveled off. After day 11, cell number decreased, whereas collagen production continued to increase. Cell number and differentiation at day 23 were independent of the timing of the mechanical stimulation applied. In conclusion, static load applied to BMSCs cultured on PLGA scaffolds allows for proliferation and differentiation, with loading during seeding yielding the most rapid response. Future research should be aimed at elucidating the biomechanical and biochemical characteristics of tissue formed by BMSCs on PLGA under mechanical stimulation.

Realization of a soft-MI electrospun scaffold fortissue engineering applications

Introduction: Anterior cruciate ligament (ACL) injuries are very common; in Germany incidence of ACL ruptures is estimated at 32 per 100 000 in the general population and in the sports community this rate more than doubles. Current gold standard for anterior cruciate lig- ament repair is reconstruction using an autograft [1]. However, this approach has shown some limitations. A new method has been her- alded by the Knee Team at the Bern University Hospital (Inselspital) and the Sonnenhof clinic called Dynamic Intraligamentary Stabilization (DIS), which keeps ACL remnants in place in order to promote biologi- cal healing and makes use of a dynamic screw system [2]. The aim of this study was to investigate the cytocompatibility of collagen patches in combination with DIS to support regeneration of the ACL. The spe- cific hypothesis we tested was whether MSCs would differentiate towards TCs in co-culture. Materials and methods: Primary Tenocytes (TCs) and human bone marrow derived mesenchymal stem cells (MSCs) were harvested from ACL removed during knee prothesis or from bone marrow aspirations (Ethical Permit 187/10). Cells were seeded on two types of three dimensional carriers currently approved for cartilage repair, Novocart (NC, B. Brown) and Chondro-Gide (CG, Geistlich). These scaffolds comprise collagen structures with interconnecting pores originally developed for seeding of chondrocytes in the case of CG. ~40k cells were seeded on punched zylindrical cores of 8 mm in O and cultured on CG or NC patches for up to 7 days. The cells were either cultured as TC only, MSC only or co-cultured in a 1:1 mix on the scaffolds and on both sides of culture inserts (PET, high density pore O 0.4 mm, BD, Fal- con) with cell-cell contact. We monitored DNA content, GAG and HOP-content, tracked the cells using DIL and DIO fluorescent dyes (Molecular Probes, Life technologies) and confocal laser scanning and SEM microscopy as well as RT-PCR of tenocyte specific markers (i.e. col 1 and 3, TNC, TNMD, SCXA&B, and markers of dedifferentiation ACAN, col2, MMP3, MMP13). Finally, H&E stain was interpreted on cryosections and SEM images of cells on the scaffold were taken. Results: ThecLSMimagesshowedcellproliferationoverthe7dayson both matrices, however, on CG there were much fewer MSCs attached than on NC. SEM images showed a roundish chondrocyte-like pheno- type of cells on CG whereas on NC the phenotype was more teno- cyte-like (Fig. 1). Gene expression of both, MSC and TC seem to confirm a more favorable environment in 3D for both patches rather than monolayer control.Hydroxyapatite (HA), [Ca10(PO4)6(OH)2], products are well-known as implantable ceramics for hard tissue reconstitution. HA is based on calcium phosphate, and its chemical composition and crystal structure are similar to the mineral component of human bones and teeth. The aim of this study was to evaluate the bioactivity of natural HA/hardystonite nanobiocomposites soaked in simulated body fluid (SBF). Novel natural HA/hardystonite nanobiocomposite was fabricated with 0 wt.%, 5 wt.%, 10 wt.%, and 15 wt.% of hardystonite in natural HA using ball mill for 20 minutes. The composite mixture was compacted in cylinder steel mould with 10 mm diameter under 20 MPa pressure. The discs pressed were soaked in cell laboratory, Falcon, containing SBF solution by 21 days. Samples weight loss and solution Ph were measured after 1, 3, 7, 14 and 21 days .Also, SBF solution Ca ion concentration were measured for solutions SBF after 21 day. X-ray diffraction (XRD), scanning electron microscopy (SEM) and EDS were performed to characterize the nanocomposite samples. ICP technique was utilized to evaluate Ca ion concentration released in solution SBF. Maximum bioactivity occurred in the sample containing 10 wt.% of hardystonite, which was probably due to two reasons; first, the maximum amorphous glassy phase amount, and second, the minimum crystallinity of nanobiocomposite.

222 INHIBITION OF PROSTAGLANDIN E2 BY CELECOXIB DECREASES GLYCOSAMINOGLYCAN RELEASE, HOWEVER DOES NOT STIMULATE REPAIR OF OSTEOARTHRITIC CARTILAGE TISSUE

Results: BAP level was higher in OA synovial fluid than in simple synovitis, and d-ROM level was slightness lower in OA than control synovitis, but this differences were not significant. BAP and d-ROM levels were not related with OA grade. After intra-articular injection of HA, BAP level was significantly increased and d-ROM level was reduced. On the other hand, BAP and d-ROM level were significantly decreased after injection of corticosteroid. Conclusions: Oxidative stress and anti-oxidative potential in synovial fluid were not significant difference between OA and simple synovitis. Our results showed that hyaluronan increase the anti-oxidant potential and decreased oxidative stress, but corticosteroid decreased both oxidative stress and the anti-oxidative stress potential.

40 OVEREXPRESSION OF hsa-miR-148A PROMOTES TYPE II COLLAGEN SYNTHESIS BY OSTEOARTHRITIC CHONDROCYTES

Purpose: MicroRNAs (miRNAs) regulate gene expression through basespecific interactions. The aim of this study was to further identify miRNAs differentially expressed in OA and to investigate the effect of one of these, miR-148, on the chondrogenic potential of chondrocytes from OA cartilge. Methods: Low-density Taqman arrays were used to identify miRNAs differentially expressed in OA and normal cartilage. OA chondrocytes were isolated from articular cartilage obtained from patients undergoing knee arthroplasty. At passage 2, OA chondrocytes from 6 donors were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. Chondrocytes were reverse-transfected during seeding at high density (1.26×106 cells per cm) on collagencoated culture inserts in a 96-wells transwell system. After 2 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I collagen (COL1A1), type II collagen (COL2A1) and matrix metallopeptidase 13 (MMP13). After 1 week, glycosaminoglycan (GAG), collagen and DNA content were determined using a DMMB, hydroxyproline and Picogreen assay, respectively. Type II collagen was analyzed at the protein level by Western blot. Results: 66 miRNAs were differentially expressed in OA cartilage compared to healthy cartilage, including miR-148a, which was downregulated 11 times in OA cartilage compared to normal cartilage. Overexpression of miR-148a had no effect on ACAN and COL1A1 gene expression levels and on the amount of GAG produced by the cells. MiR-148a overexpressed cells showed increased gene expression levels of COL2A1, while MMP13 was decreased. The increase at the mRNA level was reflected by an increase in the total amount of collagen, in particular type II collagen as shown by biochemical analyses.

263 REGENERATIVE CAPACITIES OF CAPRINE ARTICULAR CHONDROCYTES AND CHONDRONS ARE INFLUENCED BY THE MICROENVIRONMENT AND HARVEST SITE

Conclusions: In this study we tested our hypothesis that PRPr has antiinflammatory properties and influences gene expression of extracellular matrix forming and degrading proteins in an OA mimicking environment. An inflammatory environment was created through addition of IL-1 beta, one of the key players in OA pathogenesis, to chondrocytes in culture. Exposure of chondrocytes to the inflammatory cytokine IL-1 beta resulted in marked changes in the expression of genes involved in matrix formation and degradation as well as inflammation, many of which were reduced by the addition of PRPr. The inhibition of NFkB activation was found to be a possible mechanism through which PRPr exerted these effects. Revealing the relevant mechanisms and processes could improve application of PRP in a clinical setting. We consider these encouraging results for the further study of PRP as a conservative, autologous and cost-effective treatment for OA.