Increased chondrocyte seeding density has no positive effect on cartilage repair in an MPEG-PLGA scaffold

Purpose

This study investigates the effect of cell seeding density on cartilage repair in matrix-assisted chondrocyte implantation in vitro and in vivo.

Methods

In vitro: Four different cell seeding densities of human chondrocytes were seeded onto a porous methoxy-polyethylene glycol-polylactic-co-glycolic acid scaffold (MPEG-PLGA) polymer scaffold ASEED? (1.2 × 10⁶, 4.0 × 10⁶, 1.2 × 10⁷ and 2.0 × 10⁷ cells/cm³). The cartilage repair response was evaluated by relative gene expression of the chondrogenic markers sox9, collagen types I, II and X, and aggrecan, total DNA content and sulphated glycosaminoglycan synthesis. In vivo: Using a New Zealand white rabbit intercondylar osteochondral defect model, three different cell seeding densities (1.2 × 10⁶, 4.0 × 10⁶ and 1.2 × 10⁷ cells/cm³) were tested with an empty scaffold as control. The cartilage repair response was evaluated using O’Driscoll score.

Results

In vitro: A significant difference (p < 0.05) in total DNA content was found at day 2 but not at day 7. The low cell seeding densities yielded the highest GAG content (p < 0.001) at day 7. Collagen type I was highest (p < 0.01) at the lowest density at day 7. In vivo: No significant difference was found between the 4 groups.

Conclusions

No positive effect on cartilage repair was found using increased cell seeding density.

Level of evidence

Controlled experimental study, Level II.     

One-step osteochondral repair with cartilage fragments in a composite scaffold

Purpose

This study proposes a single-step therapeutic approach for osteochondral defects using autologous cartilage fragments loaded onto a scaffold composed of a hyaluronic acid (HA) derivative, human fibrin glue (FG) and autologous platelet-rich-plasma (PRP), in a rabbit model. The aim is to demonstrate the in vitro outgrowth of chondrocytes from cartilage fragments and the in vivo formation of a functional repair tissue.

Methods

In vitro: minced articular cartilage was loaded onto two different types of scaffold (paste or membrane) according to two different HA preparations (injectable HA-derivative or HA-derivative felt). In vivo: trochlear osteochondral defects were created in 50 adult rabbits, which were then assigned to 5 different treatment groups: cartilage fragments loaded onto membrane scaffolds with FG (Group 1) or without FG (Group 2); membrane scaffolds alone with FG (Group 3) or without FG (Group 4); empty defects (Group 5). Membrane scaffolds were used “in vivo” for simpler preparation and better adhesive properties. Repair processes were evaluated histologically and by immunohistochemistry at 1, 3, and 6 months.

Results

An in vitro time-dependent cell outgrowth from cartilage fragments was observed with both types of scaffolds. At 6 months, in vivo, cartilage fragment-loaded scaffolds induced significantly better repair tissue than the scaffold alone using histological scoring. Repair in Group 2 was superior to that in any of the control groups (p < 0.05).

Conclusion

Autologous cartilage fragments loaded onto an HA felt/FG/PRP-scaffold provided an efficient cell source, and allowed for an improvement of the repair process of ostechondral defects in a rabbit model. Human FG, however, hampered the rabbit healing process. These results may have clinical relevance as they show the potential of a novel one-stage repair technique for osteochondral defects.     

Cartilage repair using mesenchymal stem cell (MSC) sheet and MSCs-loaded bilayer PLGA scaffold in a rabbit model

Purpose

The integration of regenerated cartilage with surrounding native cartilage is a major challenge for the success of cartilage tissue-engineering strategies. The purpose of this study is to investigate whether incorporation of the power of mesenchymal stem cell (MSC) sheet to MSCs-loaded bilayer poly-(lactic-co-glycolic acid) (PLGA) scaffolds can improve the integration and repair of cartilage defects in a rabbit model.

Methods

Rabbit bone marrow-derived MSCs were cultured and formed cell sheet. Full-thickness cylindrical osteochondral defects (4 mm in diameter, 3 mm in depth) were created in the patellar groove of 18 New Zealand white rabbits and the osteochondral defects were treated with PLGA scaffold (n = 6), PLGA/MSCs (n = 6) or MSC sheet-encapsulated PLGA/MSCs (n = 6). After 6 and 12 weeks, the integration and tissue response were evaluated histologically.

Results

The MSC sheet-encapsulated PLGA/MCSs group showed significantly more amounts of hyaline cartilage and higher histological scores than PLGA/MSCs group and PLGA group (P < 0.05). In addition, the MSC sheet-encapsulated PLGA/MCSs group showed the best integration between the repaired cartilage and surrounding normal cartilage and subchondral bone compared to other two groups.

Conclusions

The novel method of incorporation of MSC sheet to PLGA/MCSs could enhance the ability of cartilage regeneration and integration between repair cartilage and the surrounding cartilage. Transplantation of autologous MSC sheet combined with traditional strategies or cartilage debris might provide therapeutic opportunities for improving cartilage regeneration and integration in humans.     

Failed cartilage repair for early osteoarthritis defects: a biochemical, histological and immunohistochemical analysis of the repair tissue after treatment with marrow-stimulation techniques

Purpose

To examine the entire repair tissue resulting from marrow-stimulation techniques in patients with early osteoarthritis.

Methods

The repair tissue and adjacent articular cartilage after failed marrow-stimulation techniques (microfracture and Pridie drilling) of 5 patients (47–65 years old) with cartilage defects and radiographic early osteoarthritis (Kellgren–Lawrence grading 1 and 2) was removed during total joint arthroplasty (mean time until analysis: 8.8 months), analysed by histology, polarized light microscopy, immunohistochemistry, biochemistry and by histological score systems.

Results

Macroscopic cartilage repair assessment revealed ICRS grades of II (nearly normal) and III (abnormal). Cartilage defects were mostly completely filled with a fibrocartilaginous tissue that had small and large fissures. Cartilage-specific stains of the repair tissue were more intense than the surrounding native cartilage but reduced compared with normal articular cartilage. The subchondral bone was incompletely restored. A new tidemark was absent. The repair tissue always showed positive immunoreactivity for types II and X collagen, and was sometimes positive for type I collagen. Proteoglycan contents of the repair tissue were generally higher than of the surrounding cartilage. The repair tissue was always more cellular than the adjacent articular cartilage. Histological scoring of the repair tissue revealed a mean Sellers score of 17.6 ± 3.0 and an ICRS grading of 7–9.

Conclusion

Failed marrow stimulation of articular cartilage defects in patients with early osteoarthritis is characterized by fibrocartilaginous repair. The balance of cell number to extracellular matrix is shifted towards an increased cell number in this tissue. Articular cartilage repair did not reach the quality of normal hyaline articular cartilage.

Level of evidence

IV.     

Osteochondral regeneration using a novel aragonite-hyaluronate bi-phasic scaffold in a goat model

Purpose

The objective of this study was to examine whether different mechanical modifications and/or impregnation of hyaluronic acid (HA) might enhance aragonite-based scaffold properties for the regeneration of cartilage and bone in an animal model.

Methods

Bi-phasic osteochondral scaffolds were prepared using coralline aragonite with different modifications, including 1- to 2-mm-deep drilled channels in the cartilage phase (Group 1, n = 7) or in the bone phase (Group 2, n = 8), and compared with unmodified coral cylinders (Group 3, n = 8) as well as empty control defects (Group 4, n = 4). In each group, four of the implants were impregnated with HA to the cartilage phase. Osteochondral defects (6 mm diameter, 8 mm depth) were made in medial and lateral femoral condyles of 14 goats, and the scaffolds were implanted according to a randomization chart. After 6 months, cartilage and bone regeneration were evaluated macroscopically and histologically by an external laboratory.

Results

Group 1 implants were replaced by newly formed hyaline cartilage and subchondral bone (combined histological evaluation according to the ICRS II-2010 and O’Driscoll et al. 34 ± 4 n = 7). In this group, the cartilaginous repair tissue showed a smooth contour and was well integrated into the adjacent native cartilage, with morphological evidence of hyaline cartilage as confirmed by the marked presence of proteoglycans, a marked grade of collagen type II and the absence of collagen type I. The average scores in other groups were significantly lower (Group 2 (n = 8) 28.8 ± 11, Group 3 (n = 8) 23 ± 9 and Group 4 (empty control, n = 4) 19.7 ± 15).

Conclusions

The implants with the mechanical modification and HA impregnation in the cartilage phase outperformed all other types of implant. Although native coral is an excellent material for bone repair, as a stand-alone material implant, it does not regenerate hyaline cartilage. Mechanical modification with drilled channels and impregnation of HA within the coral pores enhanced the scaffold’s cartilage regenerative potential. The modified implant shows young hyaline cartilage regeneration. This implant might be useful for the treatment of both chondral and osteochondral defects in humans.     

Anterior cruciate ligament regeneration using mesenchymal stem cells and collagen type I scaffold in a rabbit model

Purpose

The objective of this study was to determine whether using mesenchymal stem cells (MSC) seeded in a collagen type I scaffold would be sufficient to regenerate the torn anterior cruciate ligament (ACL).

Methods

Anterior cruciate ligament transection was performed on both knees in 10 New Zealand rabbits and then repaired with as follows: suture alone (suture-treated group, n = 6), suture associated with collagen type I scaffold (collagen type I scaffold-treated group, n = 8) or suture associated with autologous MSC seeded on collagen type I scaffold (MSC/collagen type I scaffold-treated group, n = 6). At 12-week post-intervention, the animals were killed and the ACLs were characterised macroscopically and histologically. Data of the 3 groups were against normal ACL (normal group, n = 10).

Results

Macroscopic observation found that in MSC/collagen type I scaffold group, 33 % of specimens showed a complete ACL regeneration, with a tissue similar to the normal ACL. Regeneration was not observed in the group treated with suture alone or associated with collagen type I scaffold without cells. In the latter, only a reparative attempt at the ends was observed. Histological analysis of the regenerated ACL showed a tissue with organised collagen and peripheric vessels.

Conclusions

These results provide evidence that the use of MSC seeded in a collagen type I scaffold in the treatment of ACL injuries is associated with an enhancement of ligament regeneration. This MSC-based technique is a potentially attractive tool for improving the treatment of ACL ruptures.     

Transplanted articular chondrocytes co-overexpressing IGF-I and FGF-2 stimulate cartilage repair in vivo

Purpose

The combination of chondrogenic factors might be necessary to adequately stimulate articular cartilage repair. In previous studies, enhanced repair was observed following transplantation of chondrocytes overexpressing human insulin-like growth factor I (IGF-I) or fibroblast growth factor 2 (FGF-2). Here, the hypothesis that co-overexpression of IGF-I and FGF-2 by transplanted articular chondrocytes enhances the early repair of cartilage defects in vivo and protects the neighbouring cartilage from degeneration was tested.

Methods

Lapine articular chondrocytes were transfected with expression plasmid vectors containing the cDNA for the Escherichia coli lacZ gene or co-transfected with the IGF-I and FGF-2 gene, encapsulated in alginate and transplanted into osteochondral defects in the knee joints of rabbits in vivo.

Results

After 3 weeks, co-overexpression of IGF-I/FGF-2 improved the macroscopic aspect of defects without affecting the synovial membrane. Immunoreactivity to type-I collagen, an indicator of fibrocartilage, was significantly lower in defects receiving IGF-I/FGF-2 implants. Importantly, combined IGF-I/FGF-2 overexpression significantly improved the histological repair score. Most remarkably, such enhanced cartilage repair was correlated with a 2.1-fold higher proteoglycan content of the repair tissue. Finally, there were less degenerative changes in the cartilage adjacent to the defects treated with IGF-I/FGF-2 implants.

Conclusion

The data demonstrate that combined gene delivery of therapeutic growth factors to cartilage defects may have value to promote cartilage repair. The results also suggest a protective effect of IGF-I/FGF-2 co-overexpression on the neighbouring articular cartilage. These findings support the concept of implementing gene transfer strategies for articular cartilage repair in a clinical setting.     

Autologous collagen-induced chondrogenesis technique (ACIC) for the treatment of chondral lesions of the talus

Purpose

Autologous collagen-induced chondrogenesis technique (ACIC) combines microfractures with the use of an injectable atelocollagen matrix that allows performing the whole cartilage repair treatment arthroscopically. The aim of this study was to evaluate the in vitro cytocompatibility of this biomaterial using human bone marrow mesenchymal stem cells and human chondrocytes. Moreover, the preliminary data of five patients affected by chondral lesion of the talus treated with the ACIC technique are shown.

Methods

Human bone marrow mesenchymal stem cells and human chondrocytes were seeded on solid and pre-solid atelocollagen scaffolds. Cell–scaffold constructs were cultured for 7 days and then prepared for histological analyses. Arthroscopic ACIC was performed in five patients affected by chondral lesions of the talus; they were clinically evaluated with AOFAS, VAS and Tegner score before and then after 6 months from surgery.

Results

In vitro results showed that both bone marrow mesenchymal stem cells and chondrocytes were able to efficiently colonize the whole construct, from the surface to the core, only when seeded on the pre-solid atelocollagen scaffold, but not on its solid form. No adverse events were observed in the patients treated with the ACIC technique; a significant improvement in VAS pain scale and in AOFAS score was found at 6 months follow up.

Conclusion

Injectable atelocollagen can be considered a feasible scaffold for cartilage repair treatment, in particular if used in its pre-solid form. ACIC leads to good clinical results in the treatment for chondral lesions of the talus even if longer follow-up and a higher number of patients are necessary to confirm these data.

Level of evidence

IV.     

Use of innovative biomimetic scaffold in the treatment for large osteochondral lesions of the knee

Purpose

Large osteochondral defects involve two different tissues characterized by different intrinsic healing capacity. Different techniques have been proposed to treat these lesions with results still under discussion. The aim of the study is to evaluate the clinical outcome of 19 patients treated with a type I collagen–hydroxyapatite nanostructural biomimetic osteochondral scaffold at minimum follow-up of 2 years.

Methods

Twenty lesions, 19 patients were treated with this scaffold implantation. The lesions size went from 4 to 8 cm² (mean size 5.2 ± 1.6 cm²). All patients were clinically evaluated using the International Repair Cartilage Society score, the Tegner Score and EQ-VAS. MRI was performed at 12 and 24 months after surgery and then every 12 months and evaluated with magnetic resonance observation of cartilage repair tissue scoring scale.

Results

The IKDC subjective score improved from a mean score of 35.7 ± 6.3 at the baseline evaluation to 67.7 ± 13.4 at 12-month follow-up (p < 0.0005). A further improvement was documented from 12 to 24 months (mean score of 72.9 ± 12.4 at 24 months) (p < 0.0005). The IKDC objective score confirmed the results. The Tegner activity score improvement was statistically significant (p < 0.0005). The EQ-VAS showed a significant improvement from 3.15 ± 1.09 to 7.35 ± 1.14 (p < 0.0005) at 2-year follow-up. The lesion’ site seems to influence the results showing a better outcome in the patients affected in the medial femoral condyle.

Conclusions

The use of the MaioRegen scaffold is a good procedure for the treatment for large osteochondral defects where other classic techniques are difficult to apply. It is an open one-step surgery with promising stable results at medium follow-up.

Level of evidence

IV.     

Cartilage repair in the knee with subchondral drilling augmented with a platelet-rich plasma-immersed polymer-based implant

Purpose

The aim of our study was to analyse the clinical and histological outcome after the treatment of focal cartilage defects in non-degenerative and degenerative knees with bone marrow stimulation and subsequent covering with a cell-free resorbable polyglycolic acid–hyaluronan (PGA-HA) implant immersed with autologous platelet-rich plasma (PRP).

Methods

Fifty-two patients (mean age 44 years) with focal chondral defects in radiologically confirmed non-degenerative or degenerative knees were subjected to subchondral drilling arthroscopically. Subsequently, defects were covered with the PGA-HA implant immersed with autologous PRP. At 2-year follow-up, the patients’ situation was assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and compared to the pre-operative situation and 3–12-month follow-up. Biopsies (n = 4) were harvested at 18–24 months after implantation and were analysed by histology and collagen type II immune staining.

Results

At 1- and 2-year follow-up, the KOOS showed clinically meaningful and significant (p < 0.05) improvement in all subcategories compared to baseline and to 3-month follow-up. There were no differences in KOOS data obtained after 2 years compared to 1 year after the treatment. Histological analysis of the biopsy tissue showed hyaline-like to hyaline cartilage repair tissue that was rich in cells with a chondrocyte morphology, proteoglycans and type II collagen.

Conclusions

Covering of focal cartilage defects with the PGA-HA implant and PRP after bone marrow stimulation improves the patients’ situation and has the potential to regenerate hyaline-like cartilage.

Level of evidence

Case series, Level IV.     

Cell-free repair of small cartilage defects in the Goettinger minipig: which defect size is possible?

Purpose

Cartilage repair of full-thickness chondral defects in the knees of Goettinger minipigs was assessed by treatment with cell-free collagen type-I gel plugs of three different sizes.

Methods

In 6 adult Goettinger minipigs, three full-thickness chondral defects were created in the trochlear groove of one knee of the hind leg. These defects were treated with a cell-free collagen type-I gel plug of 8, 10, or 12 mm diameter. All animals were allowed unlimited weight bearing. After 1 year, the animals were killed. Immediately after recovery, a non-destructive biomechanical testing was performed. The repair tissue quality was evaluated immunohistologically, collagen type-II protein was quantified, and a semiquantitative score (O’Driscoll score) was calculated.

Results

After 1 year, a high number of cells migrated into the initially cell-free collagen gel plugs and a hyaline-like repair tissue had been created. The O’Driscoll scores were: 8 mm, 21.2 (SD, 2.8); 10 mm, 21.5 (SD, 1.6); and 12 mm, 22.3 (SD, 1.0). The determination of the e-modulus, creep and relaxation revealed that mechanical properties of the two smaller defects were closer to unaffected hyaline cartilage.

Conclusions

As cell-free collagen type-I gel plugs of all three different sizes created hyaline-like repair tissue, this system seems suitable for the treatment of even larger defects.     

Early resumption of physical activities leads to inferior clinical outcomes after matrix-based autologous chondrocyte implantation in the knee

Purpose

Matrix-based autologous chondrocyte implantation is a well-established operation procedure for full cartilage defects. When to resume physical activity after matrix-based autologous chondrocyte implantation is controversial. Our hypothesis was that early resumption of physical activity leads to a worse clinical outcome after matrix-based autologous chondrocyte implantation in the knee two years post-operatively. Physical activity is defined as any kind of impact sport.

Methods

Forty-four patients with cartilage defects of the knee were treated with matrix-based autologous chondrocyte implantation (Novocart^®3D). All patients were assessed preoperatively and after a period of 24 months with the University of California Los Angeles (UCLA) Activity score. The return to physical activities or sports after matrix-based autologous chondrocyte implantation was documented. Patients were evaluated using the International Knee Documentation Committee Knee Examination Form and visual analogue scale for pain after 6, 12 and 24 months.

Results

Fifty-five percent showed an unchanged level of physical activity in the UCLA Activity score post-operatively. About 35 % showed a lower level and 10 % a higher level of physical activity. The average return to physical activities or sports after matrix-based autologous chondrocyte implantation procedure was 10.2 months. Patients with a later return of sports after 12 months showed significantly better clinical results after two years. In particular, patients who started practicing impact sport after 12 months post-operatively showed significantly better results.

Conclusion

Resuming physical activity including impact sports without waiting at least 12 months after the operation leads to inferior outcomes up to 24 months after matrix-based autologous chondrocyte implantation.

Level of evidence

Level IV.     

Nicotine-induced chondrogenic differentiation of human bone marrow stromal cells in vitro

Purpose

Nicotine has been reported that it has a dose-dependent effect on matrix mineralization by human bone marrow cells. However, there is no relevant research concerning on chondrogenic differentiation potential of bone marrow stromal stem cells (BMSCs) treated with nicotine in vitro. The aims of the study were to examine the effects of nicotine (0, 10^?7, 10^?6 and 10^?5 M) on the proliferation and chondrogenic differentiation of BMSCs from three healthy donors in vitro.

Methods

BMSCs proliferation was analyzed by CCK8 assay and real-time polymerase chain reaction was used to assay the expression of type II collagen, aggrecan, type I collagen and type X collagen. The proteoglycan content was stained by Alcian blue, and the sulfated glycosaminoglycan (sGAG) content of BMSCs was quantified spectrofluorometrically using dimethylmethylene blue.

Results

The cell viability was not significantly impaired until up to a concentration of 10^?5 M nicotine. Nicotine promoted the proliferation and enhanced the expression of type II collagen at the level up to 10^?6 M (P < 0.05). The expression of aggrecan was reduced at the concentration of 10^?5 M nicotine at day 14 (P < 0.05), and there was no significant difference in aggrecan gene expression at 10^?7 and 10^?6 M nicotine levels compared to control group (n.s.). Also the fibroblastic and hypertrophic gene expressions were down-regulated in the chondrogenic medium with 10^?7–10^?5 M nicotine (P < 0.05).

Conclusion

It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing chondrogenic differentiation capacity of BMSCs in cell-based cartilage tissue engineering. Also these results indicate that nicotine maybe a potentially useful drug for the treatment of Osteoarthritis.     

In vivo evaluation of biomechanical properties in the patellofemoral joint after matrix-associated autologous chondrocyte transplantation by means of quantitative T2 MRI

Purpose

To determine in vivo biomechanical properties of articular cartilage and cartilage repair tissue of the patella, using biochemical MRI by means of quantitative T2 mapping.

Methods

Twenty MR scans were achieved at 3T MRI, using a new 8-channel multi-function coil allowing controlled bending of the knee. Multi-echo spin-echo T2 mapping was prepared in healthy volunteers and in age- and sex-matched patients after matrix-associated autologous chondrocyte transplantation (MACT) of the patella. MRI was performed at 0° and 45° of flexion of the knee after 0 min and after 1 h. A semi-automatic region-of-interest analysis was performed for the whole patella cartilage. To allow stratification with regard to the anatomical (collagen) structure, further subregional analysis was carried out (deep–middle–superficial cartilage layer). Statistical analysis of variance was performed.

Results

During 0° flexion (decompression), full-thickness T2 values showed no significant difference between volunteers (43 ms) and patients (41 ms). Stratification was more pronounced for healthy cartilage compared to cartilage repair tissue. During 45° flexion (compression), full-thickness T2 values within volunteers were significantly increased (54 ms) compared to patients (44 ms) (p < 0.001). Again, stratification was more pronounced in volunteers compared to patients. The volunteer group showed no significant increase in T2 values measured in straight position and in bended position. There was no significant difference between the 0- and the 60-min MRI examination. T2 values in the patient group increased between the 0- and the 60-min examination. However, the increase was only significant in the superior cartilage layer of the straight position (p = 0.021).

Conclusion

During compression (at 45° flexion), healthy patellar cartilage showed a significant increase in T2-values, indicating adaptations of water content and collagen fibril orientation to mechanical load. This could not be observed within the patella cartilage after cartilage repair (MACT) of the patella, most obvious due to a lack of biomechanical adjustment.

Level of evidence

III.     

Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF

Purpose

Minced chondral fragments are becoming popular as a source of cells for cartilage repair, as a growing interest is developing towards one-stage procedures to treat cartilage lesions. The purpose of this study is to (A) compare cell outgrowth from cartilage fragments of adult and young donors using two different types of scaffolds and (B) evaluate the influence of transforming-growth-factor-β1 (TGF-β1) and granulocyte colony-stimulating factor (G-CSF) on chondrocyte behaviour.

Methods

In part (A) cartilage fragments from adult and young donors were either loaded onto an HA-derivative injectable paste scaffold or onto an HA-derivative membrane scaffold. Construct sections were then examined for cell counting after 1, 2 and 3 months. In part (B) only membrane scaffolds were prepared using cartilage fragments from young donors. Constructs were cultured either in standard growth medium or in the presence of specific growth factors, such as TGF-β1 or G-CSF or TGF-β1 + G-CSF. After 1 month, construct sections were examined for cell counting. Expression of chondrocyte markers (SOX9, CD151, CD49c) and proliferative markers (β-catenin, PCNA) was assessed using immunofluorescence techniques, both in unstimulated construct sections and in cells from unstimulated and stimulated construct cultures.

Results

Part (A): histological analysis showed age-dependent and time-dependent chondrocyte migration. A significant difference (p < 0.05) was observed between young and older donors at the same time point. No difference was detected between the two types of scaffolds within the same group at the same time point. Part (B): after 1 month, the number of migrating cells/area significantly increased due to exposure to TGF-β1 and/or G-CSF (p < 0.05). Immunofluorescence revealed that outgrowing cells from unstimulated scaffold sections were positive for SOX9, CD151, CD49c and G-CSF receptor. Immunofluorescence of cells from construct cultures showed an increase in β-catenin in all stimulated groups and an increased PCNA expression in G-CSF-exposed cultures (p < 0.05).

Conclusion

Outgrowing cells may represent a subset of chondrocytes undergoing a phenotypic shift towards a proliferative state. TGF-β1, and to a greater extent G-CSF, may accelerate this outgrowth. The clinical relevance of this study may involve a potential future clinical application of scaffolds preloaded with growth factors as an additional coating for chondral fragments. Indeed, a controlled delivery of G-CSF, widely employed in various clinical settings, might improve the repair process driven by minced human cartilage fragments during one-stage cartilage repair.     

Correlation between apparent diffusion coefficient and viscoelasticity of articular cartilage in a porcine model

Objective

Quantitative MR imaging techniques of degenerative cartilage have been reported as useful indicators of degenerative changes in cartilage extracellular matrix, which consists of proteoglycans, collagen, non-collagenous proteins, and water. Apparent diffusion coefficient (ADC) mapping of cartilage has been shown to correlate mainly with the water content of the cartilage. As the water content of the cartilage in turn correlates with its viscoelasticity, which directly affects the mechanical strength of articular cartilage, ADC can serve as a potentially useful indicator of the mechanical strength of cartilage. The aim of this study was to investigate the correlation between ADC and viscoelasticity as measured by indentation testing.

Materials and methods

Fresh porcine knee joints (n?=?20, age 6?months) were obtained from a local abattoir. ADC of porcine knee cartilage was measured using a 3-Tesla MRI. Indentation testing was performed on an electromechanical precision-controlled system, and viscosity coefficient and relaxation time were measured as additional indicators of the viscoelasticity of cartilage. The relationship between ADC and viscosity coefficient as well as that between ADC and relaxation time were assessed.

Results

ADC was correlated with relaxation time and viscosity coefficient (R²?=?0.75 and 0.69, respectively, p?Conclusions This study found a moderate correlation between ADC and viscoelasticity in the superficial articular cartilage. Both molecular diffusion and viscoelasticity were higher in weight bearing than non-weight-bearing articular cartilage areas.     

Osteochondral tissue engineering approaches for articular cartilage and subchondral bone regeneration

Purpose

Osteochondral defects (i.e., defects which affect both the articular cartilage and underlying subchondral bone) are often associated with mechanical instability of the joint and therefore with the risk of inducing osteoarthritic degenerative changes. This review addresses the current surgical treatments and most promising tissue engineering approaches for articular cartilage and subchondral bone regeneration.

Methods

The capability to repair osteochondral or bone defects remains a challenging goal for surgeons and researchers. So far, most clinical approaches have been shown to have limited capacity to treat severe lesions. Current surgical repair strategies vary according to the nature and size of the lesion and the preference of the operating surgeon. Tissue engineering has emerged as a promising alternative strategy that essentially develops viable substitutes capable of repairing or regenerating the functions of damaged tissue.

Results

An overview of novel and most promising osteochondroconductive scaffolds, osteochondroinductive signals, osteochondrogenic precursor cells, and scaffold fixation approaches are presented addressing advantages, drawbacks, and future prospectives for osteochondral regenerative medicine.

Conclusion

Tissue engineering has emerged as an excellent approach for the repair and regeneration of damaged tissue, with the potential to circumvent all the limitations of autologous and allogeneic tissue repair.

Level of evidence

Systematic review, Level III.     

T2 mapping and dGEMRIC after autologous chondrocyte implantation with a fibrin-based scaffold in the knee: Preliminary results

Objective

To assess repair tissue (RT) after the implantation of BioCart™II, an autologous chondrocyte implantation (ACI) technique with a fibrin-hyaluronan polymer as scaffold. T2 mapping and delayed Gadolinium Enhanced Magnetic Resonance Imaging of Cartilage (dGEMRIC) were used to gain first data on the biochemical properties of BioCart™II RT in vivo.

Methods

T2 mapping and dGEMRIC were performed at 3 T in five patients (six knee joints) who had undergone ACI 15-27 months before. T2 maps were obtained using a pixel wise, mono-exponential non-negative least squares fit analysis. For quantitative T1 mapping a dual flip angle 3D GRE sequence was used and T1 maps were calculated pre- and post-contrast using IDL software. Subsequent region of interest analysis was carried out in comparison with morphologic MRI.

Results

A spatial variation of T2 values in both hyaline, normal cartilage (NC) and RT was found. Mean RT T2 values and mean NC T2 values did not differ significantly. Relative T2 values were calculated from global RT and NC T2 and showed a small range (0.84-1.07). The relative delta relaxation rates (rΔR1) obtained from the T1 maps had a wider range (0.77-4.91).

Conclusion

T2 mapping and dGEMRIC provided complementary information on the biochemical properties of the repair tissue. BioCart™II apparently can provide RT similar to hyaline articular cartilage and may become a less-invasive alternative to ACI with a periosteal flap.     

Autologous chondrocyte implantation in children and adolescents

Purpose

Autologous chondrocyte implantation (ACI) is a well-established treatment method for cartilage defects in knees. Age-related grouping was based on expression data of cartilage-specific markers. Specificities of ACI in the different populations were analysed.

Methods

Two hundred and sixty-seven patients undergoing ACI in the knee between 2006 and 2010 were included in this analysis. Cell characteristics and expression data of cartilage-specific surface markers as CD44, aggrecan and collagen type II were statistically analysed for age association. Epidemiological data of the defined groups were compared. Course of treatment was evaluated using MRI.

Results

A correlation analysis showed statistically significant associations between age and aggrecan or collagen type II expression in all patients <30 years. A cluster analysis could predict age-dependent expression of these markers separating groups with an average age of 18.1 ± 2.3 and 23.6 ± 4.2 years, respectively (p < 0.02). Discriminance analysis suggested the age border between adults and juveniles at about 20 years. There was no influence of age on cell characteristics or CD44 expression. In the 19 of 267 patients with an age ≤18 years, gender distribution was not different compared to adults, but patella was significantly more affected. Cartilage lesions were mainly caused by osteochondritis dissecans (OCD) and trauma. The Knee Osteoarthritis Scoring System in MRI reached 4.8 ± 2.3 points before, declining to 3.3 ± 2.3 points 6 and 12 months after the operation.

Conclusions

Age-related expression of cartilage-specific markers allows definition of adolescents in cartilage regenerating surgery. Chondromalacia in these patients is mainly caused by OCD or trauma.

Level of evidence

Case series, Level IV.     

Nutrition and degeneration of articular cartilage

Purpose

To determine the importance of synovial fluid (SF) or subchondral bone marrow (BM) as nutrition sources in cartilage degeneration.

Methods

Ninety-five-month-old male rabbits were randomly divided into 5 groups according to sources of nutrition: SFBM-both; BM-only; SF-only; None-SFBM; and Free plug (unrestricted). Nutrition to 4-mm-diameter cylindrical osteochondral plugs created on the trochlea of the distal femurs was obstructed by Polyvinyl Chloride (PVC) cap. Cartilage changes were assessed after 4, 8, and 12 weeks by histology, immunohistochemistry, and real-time PCR.

Results

Cartilage in the BM-only group suffered the greatest damage, followed by the None-SFBM and SF-only groups. Apoptosis was increased in the BM-only and None-SFBM groups compared with others. Cartilage was significantly thinner at all time points in the BM-only and None-SFBM groups when compared with SFBM-both and Free plug, whereas in the SF-only group, this difference occurred after 8 weeks. Compared with SFBM-both and Free plug, expression of collagen II and aggrecan mRNAs in all groups was decreased but MMP-3 increased, respectively.

Conclusion

Our data indicate that SF-derived nutrition is the dominant source of sustenance for adult cartilage structure and function. Cartilage damage is observed when the only nutrition source is the BM.