Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3. Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification. Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3. Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored.

Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model.

Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function.

Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs.

Autologous matrix-induced chondrogenesis (AMIC) is a new treatment option for full-thickness cartilage defect repair using the well-known microfracturing technique combined with a porcine collagen type I/III matrix implant and partially autologous fibrin sealant.

A retrospective study has being carried out to investigate the objective and subjective clinical outcome of this procedure over a period of up to 2 years after the operation. 18 patients (10 male, 8 female) with localised cartilage defects were treated with AMIC.

The mean age was 37 13 years. Defects treated were localised retropatellar (6), on the medial femoral condyle (7), on the lateral femoral condyle (2) and multiple lesions (3). During the clinical follow-up these patients were evaluated with the help of 3 different scores (IKDC score, Cincinnati score, Lysholm-Gillquist score).

For the collective of 18 patients, one or more years had elapsed since the operation at the time this study was completed. 10 patients were included into the 2-year evaluation. The IKDC Score showed a mean improvement from 28 to 58 out of 100 at 1-year and from 25.5 to 69 out of 100 at 2-years post-operative. The Cincinnati and Lysholm-Gillquist scores showed the same tendency with an improvement of about 40 pecent at 1 year and about 55 percent at 2 years compared to pre-operative value. The improvement in the IKDC Score as well as the Cincinnati and Lysholm-Gillquist suggest that AMIC is a promising alternative in the treatment of local cartilage defects in the knee with good short and possibly mid-term results.

Further follow up will reveal, if the good results are durable and AMIC, as matrix enhanced microfracturing technique can become a valuable, recognised cartilage repair technique.

Introduction

We describe five results of a novel single stage arthroscopic technique for the treatment of articular cartilage defects of the knee. This involves micro drilling and application of Atelo-collagen (Coltrix) and fibrin gel scaffold.

Purpose

Osteochondral lesions (OCL) of the talus remain a challenging therapeutic task to orthopaedic surgeons. Several operative techniques are available for treatment, e.g. autologous chondrocyte implantation (ACI), osteochondral autograft transfer system (OATS), matrix-induced autologous chondrocyte implantation (MACI).

Good early results are reported; however, disadvantages are sacrifice of healthy cartilage of another joint or necessity of a two-stage procedure. This case describes a novel, one-step operative treatment of OCL of the talus utilizing the autologous matrix-induced chondrogenesis (AMIC) technique in combination with a collagen I/III membrane.

Chondral defects of the knee are common and often seen in young and active individuals. A novel single stage arthroscopic technique for the treatment of articular cartilage defects in the knee is described. This involves microfracture and application of concentrated bone marrow aspirate cells (BMAC) with fibrin and Hyaluronic Acid as a gel. After a representative preclinical study, the 5 year results of a prospective clinical study are presented.

The pre-clinical study involved two groups of rabbits with standardised lesions treated with microfracture alone and microfracture combined with fibrin/HA/BMAC application. New cartilage from both groups was subjected to staining with H&E for tissue morphology, toluidine blue (collagen) and safranin O (GAG), immunohistochemistry with antibodies for collagen type I and II, and scanning and transmission electron microscopy to analyse the microstructural morphologies. The fibrin/HA/BMAC group scored better than the microfracture group on all tests.

A subsequent prospective clinical study patients (n=60) with symptomatic ICRS grade III/IV chondral defects (lesion size 2–8cm2). The surgical procedure involved debridement of the lesion, micro-fracture and application of fibrin/HA/BMAC gel under CO2 insufflation. Patients underwent morphological evaluation with MRI (T2*-mapping and d-GEMRIC scans). Clinical assessment employed the Lysholm, IKDC and KOOS scores while radiological assessment was performed with MOCART score.

At 5 years, Lysholm score was 78, compared to 51 pre-operatively (p<0.05). KOOS (symptomatic) improved to 90 from 66 (p<0.05). IKDC (subjective) went to 80 from 39 (p<0.05). The mean T2* relaxation-times for the repair tissue and native cartilage were 26 and 29.9 respectively. Average MOCART score for all lesions was 70.

This technique shows encouraging clinical results at 5 year follow-up. The morphological MRI shows good cartilage defect filling and the biochemical MRI suggests hyaline like repair tissue.

Introduction

We describe a single stage arthroscopic procedure for the treatment of articular cartilage defects in the knee. The novel procedure involves microdrilling and application of atellocollagen and fibrin gel. The aim of the study was to evaluate the clinical outcomes at 4 years.