Ethics in musculoskeletal regenerative medicine; guidance in choosing the appropriate comparator in clinical trials.

BACKGROUND: Regenerative Medicine (RM) techniques aimed at the musculoskeletal system are increasingly translated to clinical trials and patient care. This revolutionary era in science raises novel ethical challenges. One of these challenges concerns the appropriate choice of the comparator in (randomized controlled) trials, including the ethically contentious use of sham procedures. To date, only general guidelines regarding the choice of the comparator exist. OBJECTIVE: To provide specific guidelines for clinical trial comparator choice in musculoskeletal RM. METHODS: In this manuscript, we discuss the ethics of comparator selection in RM trials. First, we make a classification of RM interventions according to different health states from disease prevention, return to normal health, postponing RM treatment, supplementing RM treatment, substituting RM treatment, improving RM outcome, and slowing progression. Subsequently, per objective, the accompanying ethical points to consider are evaluated with support from the available literature. RESULTS: a sham procedure is demonstrated to be an ethically acceptable comparator in RM trials with certain objectives, but less appropriate for musculoskeletal RM interventions that aim at preventing disease or substituting a surgical treatment. The latter may be compared to 'standard of care'. CONCLUSION: From a scientific perspective, choosing the correct comparator based on ethical guidelines is a step forward in the success of musculoskeletal RM.

Hyaluronic Acid-Based Hydrogel Coating Does Not Affect Bone Apposition at the Implant Surface in a Rabbit Model.

BACKGROUND: Uncemented orthopaedic implants rely on the bone-implant interface to provide stability, therefore it is essential that a coating does not interfere with the bone-forming processes occurring at the implant interface. In addition, local application of high concentrations of antibiotics for prophylaxis or treatment of infection may be toxic for osteoblasts and could impair bone growth. QUESTIONS/PURPOSES: In this animal study, we investigated the effect of a commercially available hydrogel, either unloaded or loaded with 2% vancomycin. We asked, does unloaded hydrogel or hydrogel with vancomycin (1) interfere with bone apposition and timing of bone deposition near the implant surface; and (2) induce a local or systemic inflammatory reaction as determined by inflammation around the implant and hematologic parameters. METHODS: In 18 New Zealand White rabbits, an uncoated titanium rod (n = 6), a rod coated with unloaded hydrogel (n = 6), or a rod coated with 2% vancomycin-loaded hydrogel (n = 6) was implanted in the intramedullary canal of the left tibia. After 28 days, the bone volume fraction near the implant was measured with microCT analysis, inflammation was semiquantitatively scored on histologic sections, and timing of bone apposition was followed by semiquantitative scoring of fluorochrome incorporation on histologic sections. Two observers, blinded to the treatment, scored the sections and reconciled their scores if there was a disagreement. The hematologic inflammatory reaction was analyzed by measuring total and differential leukocyte counts and erythrocyte sedimentation rates in blood. With group sizes of six animals per group, we had 79% power to detect a difference of 25% in histologic scoring for infection and inflammation. RESULTS: No differences were found in the amount of bone apposition near the implant in the No Gel group (48.65% ± 14.95%) compared with the Gel group (59.97% ± 5.02%; mean difference [MD], 11.32%; 95% CI, -3.89% to 26.53%; p = 0.16) or for the Van2 group (56.12% ± 10.06%; MD, 7.46; 95% CI, -7.75 to 22.67; p = 0.40), with the numbers available. In addition, the scores for timing of bone apposition did not differ between the No Gel group (0.50 ± 0.55) compared with the Gel group (0.33 ± 0.52; MD, -0.17; 95% CI, -0.86 to 0.53; p = 0.78) or the Van2 group (0.83 ± 0.41; MD, 0.33; 95% CI, -0.36 to 1.03; p = 0.42). Furthermore, we detected no differences in the histopathology scores for inflammation in the No Gel group (2.33 ± 1.67) compared with the Gel group (3.17 ± 1.59; MD, 0.83; 95% CI, -0.59 to 2.26; p = 0.31) or to the Van2 group (2.5 ± 1.24; MD, 0.17; 95% CI, -1.26 to 1.59; p = 0.95). Moreover, no differences in total leukocyte count, erythrocyte sedimentation rate, and neutrophil, monocyte, eosinophil, basophil, and lymphocyte counts were present between the No Gel or Van2 groups compared with the Gel control group, with the numbers available. CONCLUSION: The hydrogel coated on titanium implants, unloaded or loaded with 2% vancomycin, had no effect on the volume or timing of bone apposition near the implant, and did not induce an inflammatory reaction in vivo, with the numbers available. CLINICAL RELEVANCE: Antibiotic-loaded hydrogel may prove to be a valuable option to protect orthopaedic implants from bacterial colonization. Future clinical safety studies will need to provide more evidence that this product does not impair bone formation near the implant and prove the safety of this product.

Arthroscopic airbrush assisted cell implantation for cartilage repair in the knee: a controlled laboratory and human cadaveric study.

OBJECTIVE: The objective of this study was to investigate the feasibility of arthroscopic airbrush assisted cartilage repair. METHODS: An airbrush device (Baxter) was used to spray both human expanded osteoarthritic chondrocytes and choncrocytes with their pericellular matrix (chondrons) at 1 × 10(6) cells/ml fibrin glue (Tissucol, Baxter) in vitro. Depth-dependent cell viability was assessed for both methods with confocal microscopy. Constructs were cultured for 21 days to assess matrix production. A controlled human cadaveric study (n = 8) was performed to test the feasibility of the procedure in which defects were filled with either arthroscopic airbrushing or needle extrusion. All knees were subjected to 60 min of continuous passive motion and scored on outline attachment and defect filling. RESULTS: Spraying both chondrocytes and chondrons in fibrin glue resulted in a homogenous cell distribution throughout the scaffold. No difference in viability or matrix production between application methods was found nor between chondrons and chondrocytes. The cadaveric study revealed that airbrushing was highly feasible, and that defect filling through needle extrusion was more difficult to perform based on fibrin glue adhesion and gravity-induced seepage. Defect outline and coverage scores were consistently higher for extrusion, albeit not statistically significant. CONCLUSION: Both chondrons and chondrocytes can be evenly distributed in a sprayed fibrin glue scaffold without affecting viability while supporting matrix production. The airbrush technology is feasible, easier to perform than needle extrusion and allows for reproducible arthroscopic filling of cartilage defects.

Overexpression of hsa-miR-148a promotes cartilage production and inhibits cartilage degradation by osteoarthritic chondrocytes.

OBJECTIVE: Hsa-miR-148a expression is decreased in Osteoarthritis (OA) cartilage, but its functional role in cartilage has never been studied. Therefore, our aim was to investigate the effects of overexpressing hsa-miR-148a on cartilage metabolism of OA chondrocytes. DESIGN: OA chondrocytes were transfected with a miRNA precursor for hsa-miR-148a or a miRNA precursor negative control. After 3, 7, 14 and 21 days, real-time PCR was performed to examine gene expression levels of aggrecan (ACAN), type I, II, and X collagen (COL1A1, COL2A1, COl10A1), matrix metallopeptidase 13 (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and the serpin peptidase inhibitor, clade H (heat shock protein 47), member 1 (SERPINH1). After 3 weeks, DNA content and proteoglycan and collagen content and release were determined. Type II collagen was analyzed at the protein level by Western blot. RESULTS: Overexpression of hsa-miR-148a had no effect on ACAN, COL1A1 and SERPINH1 gene expression, but increased COL2A1 and decreased COL10A1, MMP13 and ADAMTS5 gene expression. Luciferase reporter assay confirmed direct interaction of miR-148a and COL10A1, MMP13 and ADAMTS5. The matrix deposited by the miR-148a overexpressing cells contained more proteoglycans and collagen, in particular type II collagen. Proteoglycan and collagen release into the culture medium was inhibited, but total collagen production was increased. CONCLUSION: Overexpression of hsa-miR-148a inhibits hypertrophic differentiation and increases the production and deposition of type II collagen by OA chondrocytes, which is accompanied by an increased retention of proteoglycans. Hsa-miR-148a might be a potential disease-modifying compound in OA, as it promotes hyaline cartilage production.

Enhanced cell-induced articular cartilage regeneration by chondrons; the influence of joint damage and harvest site.

OBJECTIVE: Interactions between chondrocytes and their native pericellular matrix provide optimal circumstances for regeneration of cartilage. However, cartilage diseases such as osteoarthritis change the pericellular matrix, causing doubt to them as a cell source for autologous cell therapy. METHODS: Chondrons and chondrocytes were isolated from stifle joints of goats in which cartilage damage was surgically induced in the right knee. After 4 weeks of regeneration culture, DNA content and proteoglycan and collagen content and release were determined. RESULTS: The cartilage regenerated by chondrons isolated from the damaged joint contained less proteoglycans and collagen compared to chondrons from the same harvest site in the nonoperated knee (P < 0.01). Besides, chondrons still reflected whether they were isolated from a damaged joint, even if they where isolated from the opposing or adjacent condyle. Although chondrocytes did not reflect this diseased status of the joint, chondrons always outperformed chondrocytes, even when isolated from the damaged joints (P < 0.0001). Besides increased cartilage production, the chondrons showed less collagenase activity compared to the chondrocytes. CONCLUSION: Chondrons still outperform chondrocytes when they were isolated from a damaged joint and they might be a superior cell source for articular cartilage repair and cell-induced cartilage formation.

One-stage focal cartilage defect treatment with bone marrow mononuclear cells and chondrocytes leads to better macroscopic cartilage regeneration compared to microfracture in goats.

OBJECTIVE: The combination of chondrocytes and mononuclear fraction (MNF) cells might solve the expansion induced dedifferentiation problem of reimplanted cells in autologous chondrocytes implantation as sufficient cells would be available for direct, one-stage, implantation. Earlier in vitro work already showed a positive stimulation of cartilage specific matrix production when chondrocytes and MNF cells were combined. Therefore, this study aimed to evaluate cartilage regeneration using a one-stage procedure combining MNF cells and primary chondrocytes for the treatment of focal cartilage lesions in goats compared to microfracture treatment. DESIGN: Freshly created focal cartilage defects were treated with either a combination of chondrocytes and MNF cells embedded in fibrin glue or microfracture treatment. After 6 months follow-up local regeneration as well as the general joint cartilage health were evaluated using validated scores and biochemical assays. RESULTS: Macroscopic (P = 0.015) scores for the cartilage surface at the treated defect were, after 6 months, significantly higher for the chondrocyteMNF treatment compared to microfracture-treated defects, but microscopic scores were not (P = 0.067). The articulating cartilage showed more (P = 0.005) degeneration following microfracture treatment compared to chondrocyteMNF treatment. Biochemical glycosaminoglycans (GAG) evaluation did not reveal differences between the treatments. Both treatments had resulted in a slight to moderate cartilage degeneration at other locations in the joint. CONCLUSION: In conclusion, treatment of focal articular cartilage lesions in goats using a combination of MNF cells from bone marrow and unexpanded chondrocytes leads to better macroscopic regeneration compared to microfracture, however needs further fine-tuning to decrease the negative influence on other joint compartments.

Delayed gadolinium enhanced MRI of cartilage (dGEMRIC) can be effectively applied for longitudinal cohort evaluation of articular cartilage regeneration.

OBJECTIVE: Delayed gadolinium enhanced magnetic resonance imaging (MRI) of cartilage (dGEMRIC) facilitates non-invasive evaluation of the glycosaminoglycan content in articular cartilage. The primary aim of this study was to show that the dGEMRIC technique is able to monitor cartilage repair following regenerative cartilage treatment. DESIGN: Thirty-one patients with a focal cartilage lesion underwent a dGEMRIC scan prior to cartilage repair surgery and at 3 and 12 months follow-up. At similar time points clinical improvement was monitored using the Knee injury and Osteoarthritis Outcome Score (KOOS) and Lysholm questionnaires. Per MRI scan several regions-of-interest (ROIs) were defined for different locations in the joint. The dGEMRIC index (T1gd) was calculated for each ROI. Repeated-measures analysis of variance (RMANOVA) analysis was used to evaluate improvement in clinical scores and MRI T1gd over time. Also regression analysis was performed to show the influence of local repair on cartilage quality at distant locations in the knee. RESULTS: Clinical scores and the dGEMRIC T1gd per ROI showed a statistically significant improvement (P < 0.01), from baseline, at 12 months follow-up. Also, improvement from baseline in T1gd of the ROI defining the treated cartilage defect showed a direct relationship (P < 0.007) to the improvement of the T1gd of ROI at other locations in the joint. CONCLUSIONS: The dGEMRIC MRI protocol is a useful method to evaluate cartilage repair. In addition, local cartilage repair influenced the cartilage quality at other location in the joint. These findings validate the use of dGEMRIC for non-invasive evaluation of the effects of cartilage regeneration.

A validated new histological classification for intervertebral disc degeneration.

UNLABELLED: Histology is an important outcome variable in basic science and pre-clinical studies regarding intervertebral disc degeneration (IVD). Nevertheless, an adequately validated histological classification for IVD degeneration is still lacking and the existing classifications are difficult to use for inexperienced observers. OBJECTIVE: Therefore the aim of this study was to develop and to validate a new histological classification for IVD degeneration. Moreover, the new classification was compared to the frequently used non-validated classification. METHODS: The new classification was applied to human IVD sections. The sections were scored twice by two independent inexperienced observers, twice by two experienced IVD researchers and once by a pathologist. For comparison, the sections were also scored according to the classification described by Boos et al. by two experienced IVD researchers. Macroscopic grading according Thompson et al., glycosaminoglycan (GAG) content and age were used for validation. RESULTS: The new classification had an excellent intra- and a good inter-observer reliability. Intraclass Correlation Coefficients (ICC) were 0.83 and 0.74, respectively. Intra- and inter-observer reliability were comparable for experienced and inexperienced observers. Statistically significant correlations were found between the new classification, macroscopic score, GAG content in the nucleus pulposus (NP) and age; Correlation coefficient (CC) 0.79, -0.62 and 0.68, respectively. The CCs of the Boos classification were all lower compared to the new classification. CONCLUSION: the new histological classification for IVD degeneration is a valid instrument for evaluating IVD degeneration in human IVD sections and is suitable for inexperienced and experienced researchers.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles.

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

Micro-CT quantification of subchondral endplate changes in intervertebral disc degeneration.

BACKGROUND: The intervertebral disc (IVD) is dependent on nutrient provision through a cartilage layer with underlying subchondral bone, analogous to joint cartilage. In the joint, subchondral bone remodeling has been associated with osteoarthritis (OA) progression due to compromised nutrient and gas diffusion and reduced structural support of the overlaying cartilage. However, subchondral bone changes in IVD degeneration have never been quantified before. OBJECTIVE: The aim of this study is to determine the subchondral bone changes at different stages of IVD degeneration by micro-CT. METHODS: Twenty-seven IVDs including the adjacent vertebral endplates were obtained at autopsy. Midsagittal slices, graded according the Thompson score, were scanned. Per scan 12 standardized cylindrical volumes of interest (VOI) were selected. Six VOIs contained the bony endplate and trabeculae (endplate VOIs) and six accompanying VOIs only contained trabecular bone (vertebral VOIs). Bone volume as percentage of the total volume (BV/TV) of the VOI, trabecular thickness (TrTh) and connectivity density (CD) were determined. RESULTS: An increase in BV/TV and TrTh was found in endplate VOIs of IVDs with higher Thompson score whereas these values remained stable or decreased in the vertebral VOIs. CONCLUSION: The increase in bone volume combined with the increase in TrTh in endplate VOIs strongly suggest that the subchondral endplate condenses to a more dense structure in degenerated IVDs. This may negatively influence the diffusion and nutrition of the IVD. The endplate differences between intact and mild degenerative IVDs (grade II) indicate an early association of subchondral endplate changes with IVD degeneration.

Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo.

Bone regeneration is one of the major focus points in the field of regenerative medicine. A well-known stimulus of bone formation is bone morphogenetic protein-2 (BMP-2), which has already been extensively used in clinical applications. We investigated the possibility of achieving osteogenic differentiation both in vitro and in vivo as a result of prolonged presence of BMP-2 using plasmid DNA-based gene therapy. By delivering BMP-2 cDNA in an alginate hydrogel, a versatile formulation is developed. High transfection efficiencies of up to 95% were obtained in both human multipotent stromal cells (MSCs) and MG-63 cells using naked DNA in vitro. Over a period of 5 weeks, an increasing amount of biologically active BMP-2 was released from the cells and remained present in the gel. In vivo, transfected cells were found after both two and six weeks implantation in naked mice, even in groups without seeded cells, thus indicating in vivo transfection of endogenous cells. The protein levels were effective in inducing osteogenic differentiation in vitro, as seen by elevated alkaline phosphatase (ALP) production and in vivo, as demonstrated by the production of collagen I and osteocalcin in a mineralised alginate matrix. We conclude that BMP-2 cDNA incorporated in alginate hydrogel appears to be a promising new strategy for minimal-invasive delivery of growth factors in bone regeneration.

Mathematical modelling of tissue formation in chondrocyte filter cultures.

In the field of cartilage tissue engineering, filter cultures are a frequently used three-dimensional differentiation model. However, understanding of the governing processes of in vitro growth and development of tissue in these models is limited. Therefore, this study aimed to further characterise these processes by means of an approach combining both experimental and applied mathematical methods. A mathematical model was constructed, consisting of partial differential equations predicting the distribution of cells and glycosaminoglycans (GAGs), as well as the overall thickness of the tissue. Experimental data was collected to allow comparison with the predictions of the simulation and refinement of the initial models. Healthy mature equine chondrocytes were expanded and subsequently seeded on collagen-coated filters and cultured for up to 7 weeks. Resulting samples were characterised biochemically, as well as histologically. The simulations showed a good representation of the experimentally obtained cell and matrix distribution within the cultures. The mathematical results indicate that the experimental GAG and cell distribution is critically dependent on the rate at which the cell differentiation process takes place, which has important implications for interpreting experimental results. This study demonstrates that large regions of the tissue are inactive in terms of proliferation and growth of the layer. In particular, this would imply that higher seeding densities will not significantly affect the growth rate. A simple mathematical model was developed to predict the observed experimental data and enable interpretation of the principal underlying mechanisms controlling growth-related changes in tissue composition.

Scaffolds with a standardized macro-architecture fabricated from several calcium phosphate ceramics using an indirect rapid prototyping technique.

Calcium phosphate ceramics, commonly applied as bone graft substitutes, are a natural choice of scaffolding material for bone tissue engineering. Evidence shows that the chemical composition, macroporosity and microporosity of these ceramics influences their behavior as bone graft substitutes and bone tissue engineering scaffolds but little has been done to optimize these parameters. One method of optimization is to place focus on a particular parameter by normalizing the influence, as much as possible, of confounding parameters. This is difficult to accomplish with traditional fabrication techniques. In this study we describe a design based rapid prototyping method of manufacturing scaffolds with virtually identical macroporous architectures from different calcium phosphate ceramic compositions. Beta-tricalcium phosphate, hydroxyapatite (at two sintering temperatures) and biphasic calcium phosphate scaffolds were manufactured. The macro- and micro-architectures of the scaffolds were characterized as well as the influence of the manufacturing method on the chemistries of the calcium phosphate compositions. The structural characteristics of the resulting scaffolds were remarkably similar. The manufacturing process had little influence on the composition of the materials except for the consistent but small addition of, or increase in, a beta-tricalcium phosphate phase. Among other applications, scaffolds produced by the method described provide a means of examining the influence of different calcium phosphate compositions while confidently excluding the influence of the macroporous structure of the scaffolds.

Evaluation of histological scoring systems for tissue-engineered, repaired and osteoarthritic cartilage.

OBJECTIVE: Regeneration of hyaline cartilage has been the focus of an increasing number of research groups around the world. One of the most important outcome measures in evaluation of its success is the histological quality of cartilaginous tissue. Currently, a variety of histological scoring systems is used to describe the quality of osteoarthritic, in vivo repaired or in vitro engineered tissue. This review aims to provide an overview of past and currently used histological scoring systems, in an effort to aid cartilage researchers in choosing adequate and validated cartilage histological scoring systems. METHODS: Histological scoring systems for analysis of osteoarthritic, tissue engineered and in vivo repaired cartilage were reviewed. The chronological development as well as the validity and practical applicability of the scoring systems is evaluated. RESULTS: The Histological-Histochemical Grading System (HHGS) or a HHGS-related score is most often used for evaluation of osteoarthritic cartilage, however the Osteoarthritis Research Society International (OARSI) Osteoarthritis Cartilage Histopathology Assessment System seems a valid alternative. The O'Driscoll score and the International Cartilage Repair Society (ICRS) II score may be used for in vivo repaired cartilage. The 'Bern score' seems most adequate for evaluation of in vitro engineered cartilage. CONCLUSION: A great variety of histological scoring systems exists for analysis of osteoarthritic or normal, in vivo repaired or tissue-engineered cartilage, but only few have been validated. Use of these validated scores may considerably improve exchange of information necessary for advances in the field of cartilage regeneration.

Hypertrophic differentiation and calcification during intervertebral disc degeneration.

BACKGROUND: In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. OBJECTIVE: The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. METHODS: IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. RESULTS: Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. CONCLUSION: This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.

Quality of scaffold fixation in a human cadaver knee model.

OBJECTIVE: Newly developed regenerative cartilage interventions based on the application of 3D-scaffolds require a further evaluation of the surgical techniques involved. The present study compared four different scaffold fixation techniques [fibrin glue (FG), transosseous (TS) fixation, biodegradable pin (BP) fixation and continuous cartilage sutures (CS)] to implant a custom-printed porous PEOT/PBT1000/70/30 scaffold in a human cadaver knee model. METHODS: After implantation, the knees were subjected to a vertically oriented loaded continuous passive motion (CPM) protocol. The fixation techniques were evaluated after 60 and a subsequent 150 motion cycles, focusing on area coverage, outline attachment and scaffold integrity. After the total of 210 cycles, also an endpoint fixation test was performed. RESULTS: The fixation techniques revealed marginal differences for area coverage and outline attachment after 60 and 150 cycles. The FG scored higher on scaffold integrity compared to TS (P<0.05) and CS (P=0.01). Endpoint fixation was highest for the CS, whereas FG showed a weak final fixation strength (P=0.01). CONCLUSIONS: This study showed that optimal fixation cannot be combined always with high scaffold integrity. Special attention devoted to scaffold properties in relation to the fixation technique may result in an improvement of scaffold fixation, and thus clinical cartilage regenerative approaches involving these scaffolds.

Cartilage degeneration in the goat knee caused by treating localized cartilage defects with metal implants.

OBJECTIVE: The purpose of the current study was to investigate the feasibility of applying defect-size femoral implants for the treatment of localized cartilage defects in a 1-year follow-up model. METHODS: In 13 goats, a medial femoral condyle defect was created in both knees. Defects were randomly treated by immediate placement of an oxidized zirconium (OxZr) (n=9) or cobalt-chromium (CoCr) implant (n=9) or left untreated (n=8). Six un-operated knee joints served as a control. Animals were sacrificed at 52 weeks. Joints were evaluated macroscopically. Cartilage quality was analyzed macroscopically and microscopically and cartilage repair of untreated defects was scored microscopically. Glycosaminoglycan (GAG) content, release and synthesis were measured in tissue and medium. Implant osseointegration was measured by automated histomorphometry. RESULTS: Cartilage repair score of the defects was 13.3+/-3.0 out of 24 points (0=no repair, 24=maximal repair). Articular evaluation scores decreased (indicative of degeneration) in untreated defects and in defects treated with either implant (P<0.05). Macroscopical, microscopical and biochemical analysis showed that the presence of untreated defects and the implants caused considerable degeneration of medial tibial plateau, and to a lesser extent of the lateral compartment. Mean bone-implant contact was extensive and not different between materials (39.5+/-28.1% for OxZr and 42.3+/-31.5% for CoCr) (P=0.873). CONCLUSIONS: Considerable cartilage degeneration was induced in the articulating cartilage of the medial tibial plateau 1 year after creating an osteochondral defect in the medial femoral condyle. Treating this defect with a small metal implant, made of either OxZr or CoCr, could not prevent this degeneration. Further optimization of defect-size implants and their placement is required to make this the therapy of choice for the treatment of local cartilage defects.

Formalin fixation affects equilibrium partitioning of an ionic contrast agent-microcomputed tomography (EPIC-µCT) imaging of osteochondral samples.

OBJECTIVE: Equilibrium Partitioning of an Ionic Contrast agent with microcomputed tomography (EPIC-µCT) is a non-invasive technique to quantify and visualize the three-dimensional distribution of glycosaminoglycans (GAGs) in fresh cartilage tissue. However, it is unclear whether this technique is applicable to already fixed tissues. Therefore, this study aimed at investigating whether formalin fixation of bovine cartilage affects X-ray attenuation, and thus the interpretation of EPIC-µCT data. DESIGN: Osteochondral samples (n=24) were incubated with ioxaglate, an ionic contrast agent, for 22h prior to µCT scanning. The samples were scanned in both formalin-fixed and fresh conditions. GAG content was measured using a biochemical assay and normalized to wet weight, dry weight, and water content to determine potential reasons for differences in X-ray attenuation. RESULTS: The expected zonal distribution of contrast agent/GAGs was observed for both fixed and fresh cartilage specimens. However, despite no significant differences in GAG concentrations or physical properties between fixed and fresh samples, the average attenuation levels of formalin-fixed cartilage were 14.3% lower than in fresh samples. CONCLUSIONS: EPIC-µCT is useful for three-dimensional visualization of GAGs in formalin-fixed cartilage. However, a significant reduction in X-ray attenuation for fixed (compared to fresh) cartilage must be taken into account and adjusted for accordingly when quantifying GAG concentrations using EPIC-µCT.

The presence of extracellular matrix degrading metalloproteinases during fetal development of the intervertebral disc.

Matrix metalloproteinases (MMPs) regulate connective tissue architecture and cell migration through extracellular matrix (ECM) degradation and are associated with both physiological and pathological processes. Although they are known to play a role in skeletal development, little is known about the role of MMPs in intervertebral disc (IVD) development. Sixteen fetal human lumbar spine segments, obtained at autopsy, were compared with five normal, non-fetal L4-L5 IVDs. Intensity and/or localization of immunohistochemical staining for MMP-1, -2, -3 and -14 were evaluated by three independent observers. MMP-2 production and activation was quantified by gelatin zymography. MMP-1 and -14 were abundantly present in the nucleus pulposus (NP) and notochordal (NC) cells of the fetal IVDs. In non-fetal IVDs, MMP-1 and -14 staining was significantly less intense (p = 0.001 and p < 0.001, respectively). MMP-3 was found in almost the entire IVD with no significant difference from non-fetal IVDs. MMP-2 staining in the NC and NP cells of the fetal IVD was moderate, but weak in the non-fetal IVD. Gelatin zymography showed a negative correlation of age with MMP-2 activity (p < 0.001). MMP-14 immunostaining correlated positively with MMP-2 activity (p = 0.001). For the first time, the presence of MMP-1, -2, -3 and -14 in the fetal human IVD is shown and the high levels of MMP-1, -2 and -14 suggest a role in the development of the IVD. In particular, the gradual decrease in MMP-2 activation during gestation pinpoints this enzyme as key player in fetal development, possibly through activation by MMP-1 and -14.

The dog as an animal model for DISH?

Diffuse idiopathic skeletal hyperostosis (DISH) is a systemic disorder of the axial and peripheral skeleton in humans and has incidentally been described in dogs. The aims of this retrospective radiographic cohort study were to determine the prevalence of DISH in an outpatient population of skeletally mature dogs and to investigate if dogs can be used as an animal model for DISH. The overall prevalence of canine DISH was 3.8% (78/2041). The prevalence of DISH increased with age and was more frequent in male dogs, similar to findings in human studies. In the Boxer breed the prevalence of DISH was 40.6% (28/69). Dog breeds represent closed gene pools with a high degree of familiar relationship and the high prevalence in the Boxer may be indicative of a genetic origin of DISH. It is concluded that the Boxer breed may serve as an animal model for DISH in humans.