The present study analysed the clinical outcome and the histological characteristics of membrane-seeded autolo-gous chondrocytes implantation at 24 month after surgery for chondral defects.

A prospectic study was performed on fifteen patients (8 males and 7 females, mean age 38 years) suffering from cartilage lesions of the knee (12 cases) and the ankle (3 case). The patients underwent matrix-induced autologous chondrocyte implantation (MACI). Clinical outcomes were assessed by revised IKDC form and Knee Osteoarthritis and Injury Outcome Score (KOOS). At 12 months after implantation biopsy samples were obtained from 7 patients. The specimens were analysed by histochemistry, immunohistochemistry (ICRS visual histological assessment scale) and histomorphometry (Quantimet 500+).

Improvement 12 months after operation was found subjectively (39.7 to 57.9) and in articular function levels. IKDC scores showed marked improvement at 12 months (88% A/B). 90% of biopsies showed: smooth articular surface, hyaline-like matrix, columnar cell distribution, viable cells, normal subchondral bone, tide-mark. All sections were clearly stained with safranin-O, alcian blue, and revealed immunoreaction for S-100 protein, chondroitin-S and type II collagen.

Clinical improvement and hyaline-like appearance of the repair tissue indicate that MACI implantation is an effective technique for the treatment of cartilage lesions.

Introduction: The present study analysed the clinical outcome and the histological characteristics of MACI implantation at 3 years follow up.

Materials and methods: Seventeen patients (11 males and 6 females, mean age 37 years) suffering from large cartilage lesions (2cm.2) of the knee (13 cases) and the ankle (4 cases), underwent autologous chondrocyte implantation procedure in which the expanded cells were seeded on type I/III collagen membrane before transplantation (MACI – Verigen, D). Clinical outcomes were assessed by ICRS evaluation package: revised IKDC form and Knee Osteoarthritis and Injury Outcome Score (KOOS). At least 12 months after implantation biopsy samples were arthroscopically obtained from 8 patients previous informed consent. The regenerated tissue were taken according to the ICRS standardized procedure. The specimens were stained with safranin-O and alcian blue, polyclonal antibodies anti S-100 protein and monoclonal antibodies anti chondroitin sulphate, anti-collagen type I and II. The specimens were evaluated by the ICRS visual histological assessment scale.

Results: Improvement 12 months after operation was found subjectively (39.7 to 57.9) and in knee function levels. The International Knee Documentation Committee (IKDC) scores showed marked improvement at 12 months (87% A/B). 90% of biopsies showed: smooth articular surface (I:3), hyaline-like matrix cartilage (II:3), cell distribution (columnar-clusters III:2), predominantly viable cells (IV:3), normal subchondral bone (V:3), normal cartilage mineralization and tide-mark (VI:3). All sections were clearly stained with safranin-O and alcian blue. In all the specimens the cells revealed a strong immunoreaction for S-100 protein and showed a positive reaction for chondroitin-S and type II collagen. Type I collagen was immuno-detected in the more superficial layers of the biopsies. TEM analysis revealed a defined chondral cell phenotype within a chondroid matrix. Tissue heterogeneity and irregularities of the surface were observed in four cases.

Conclusions: Clinical improvement and hyaline-like appearance of the repair tissue indicate that MACI implantation is an effective technique for the treatment of large lesions of the articular cartilage of the knee and the ankle.

Tissue engineering in the treatment of cartilage lesions utilises chondrocytes or mesenchymal stem cells (MSCs) seeded on tridimensional scaffolds. These methods are associated with high costs and two surgical procedures. Aim of this study was to evaluate the healing process of osteochondral lesions treated by drilling (in order to permit the migration of MSCs) and collagen membrane implant (to facilitate cell proliferation and differentiation).

Bilateral ostechondral lesions of the knee were induced in 24 sheep: two lesions 5 mm in diameter at the femoral condyle and two at the throclear groove in each knee. The lesions were treated as follows: drilling and typeI/II collagen membrane implant. Control lesions were treated by drilling alone. Macroscopic, histoimmunohistochemical and histomorphometric analyses were performed at 1, 6 and 12 months after operation.

The treated lesions were repaired with a hyaline-like tissue at 12 months, in comparison to control lesions which showed incomplete filling with fibrous tissue.

The use of collagen membranes covering articular defects avoids cell dispersion and maintains the necessary tissue permeability. Chondrocytes or MSCs seeded on these membranes proliferate and express differentiated phenotypes. The present study showed in an experimental model that drilling and collagen membrane implant represents a surgical approach to osteochondral lesions which produces a hyaline-like scar tissue.

The search for bone substitutes has stimulated the study of growth factors with osteoinductive properties. Bone morphogenetic proteins (BMPs) have been shown to have a central role in endochondral and intramembranous bone formation and are thought to promote normal bone healing. Recent studies demonstrated that platelet-rich plasma (PRP) can provide several growth factors and stimulate osteogenesis. The aim of the present study was to analyse the in vitro effects of rhBMP-7 and PRP on phenotype and proliferation of cells from the site of non-union and from non-affected bone.

During the surgical treatment of seven cases of non-union, normal cancellous bone and tissue from the non-union site were harvested. Osteoblast-like cells and fibroblast-like cells were isolated and characterised. Mesenchymal cells were obtained from bone marrow of the same patients. Each cell type was incubated with rhBMP-7 and PRP at different concentrations. Proliferation rate and alkaline phosphatase activity were assessed at 3, 7, 15 and 30 days. Histochemical and immunohistochemical analyses were performed at 15 and 30 days.

The proliferation rate of osteoblast-like cells and mesenchymal cells wasalways higher than that of fibroblast-like cells from the non-union site. Growth factors induced mesenchymal cells to express osteoblast phenotype markers.

The results suggest that fibroblast-like cells from the site of non-union are poorly responsive to growth factors, even at highest stimulation. In surgical practice these data strongly suggest adding osteoblast-like cells and mesenchymal cells from non-affected sites at the non-union site to enhance the osteogenic response to growth factors.

The purpose of this work was to create an in vitro model of tissue-engineered cartilage structure produced by isolated swine articular chondrocytes, expanded in culture and seeded onto a biological scaffold.

Swine articular chondrocytes were enzymatically isolated from pig joints and expanded in monolayer culture. When confluence was reached, cells were resuspended and seeded in vitro onto biological collagen scaffolds for 3, 4 and 6 weeks. Samples were retrieved from the culture and analysed macroscopically and biomechanically by compressive test. Gross evaluation was performed by simple probing, sizing and weighing the samples at all time periods. A baseline of the values was also recorded at time 0. Then, samples were biomechanically tested by unconfined compression and shear tests. Finally, the samples were fixed in 4% paraformaldehyde and processed for histological evaluation. Some samples were stained with Safranin-o, and some others subjected to immunostaining analysis for type II collagen.

Upon retrieval, samples showed dimensional enlargement and mass increase over time and gross mechanic integrity by simple probing. A biomechanical test demonstrated an initial reduction in the values of compressive and shear parameters, followed by a consistent increase throughout the tested time points. Histology showed cartilage-like tissue maturing over time within the biological scaffold.

The results from this study demonstrate that isolated chondrocytes could be seeded onto a biological collagen scaffold, producing cartilage-like matrix with tissue-specific morphology and biomechanical integrity. This tissue-engineered cartilage structure is easily reproducible and it could represent a valuable model for studying the behaviour of different variables on the newly formed cartilage.

The present investigation was undertaken to explore the possible association between lower limb torsional abnormalities and some disorders of the knee, such as patellofemoral malalignment and Osgood-Schlatter disease.

Four groups of patients were subjected to clinical, radiographic and CT evaluation: 20 male and 20 female asymptomatic subjects, 27 girls affected with patellofemoral malalignment and 21 boys affected with Osgood-Schlatter disease. With CT femoral anteversion, patellar congruence angle, patellar tilt angle, condylomalleolar angle, the distance between the anterior tibial tuberosity and the trochlear groove and external tibial rotation angle could be measured. Statistical analysis was carried out by ANOVA and Student’s t-test.

In the patellofemoral malalignment group, the femoral anteversion and rotation were significantly greater than in comparison the other symptomatic or control groups. In the Osgood-Schlatter group the condylomalleolar angle and tibial rotation angle were higher than in controls.

Several authors have demonstrated the influence of changes in the torsional alignment of the leg on the genesis of many disorders of the knee. The present CT study, employing a method that is the most accurate to measure lower limb rotation, documents a close association between patellofemoral malalignment and femoral rotation and between Osgood-Schlatter disease and increased external tibial torsion. These associations does not imply a cause-effect relationship; nevertheless, it is conceivable that these torsional abnormalities, probably in conjunction with other factors, can be predisposing mechanical factors for the onset of anterior knee pain related to patellofemoral malalignment or Osgood-Schlatter disease.

Aims: Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated the clinical efficacy of autologous chondrocytes implantation in cartilage defects, but there is little information on the use of a solid scaffold and on the composition of the repair tissue. The present study analysed the clinical outcome and the histological characteristics of membrane-seeded autologous chondrocyte implantation at 12–24 month after operation.

Materials and methods: Eleven patients (7 males and 4 females, mean age 37 years) suffering from cartilage lesions of the knee (10 cases) and the ankle (1 case), underwent autologous chondrocyte implantation procedure in which the expanded cells were seeded on type I/III collagen membrane before transplantation (MACI – Verigen, D). Clinical outcomes were assessed by ICRS evaluation package: revised IKDC form and Knee Osteoarthritis and Injury Outcome Score (KOOS). At least 12 months after implantation biopsy samples were arthroscopically obtained from 7 patients previous informed consent. The regenerated tissue were taken according to the ICRS standardized procedure. The specimens were stained with safranin-O and alcian blue, polyclonal antibodies anti S-100 protein and monoclonal antibodies anti chondroitin sulphate, anti-collagen type I and II. Moreover the number of cells/area was quantitatively assessed by histomorphometric method (Quantimet 500+). Ultrastructural analysis was also performed by transmission electron microscopy (TEM). The specimens were evaluated by the ICRS visual histological assessment scale.

Results: Improvement 12 months after operation was found subjectively (39.7 to 57.9) and in knee function levels. The International Knee Documentation Committee (IKDC) scores showed marked improvement at 12 months (87% A/B). 90% of biopsies showed: smooth articular surface (I:3), hyaline-like matrix cartilage (II:3), cell distribution (columnar-clusters III:2), predominantly viable cells (IV:3), normal subchondral bone (V:3), normal cartilage mineralization and tide-mark (VI:3). All sections were clearly stained with safranin-O and alcian blue. In all the specimens the cells revealed a strong immunoreaction for S-100 protein and showed a positive reaction for chondroitin-S and type II collagen. Type I collagen was immuno-detected in the more superficial layers of the biopsies. TEM analysis revealed a defined chondral cell phenotype within a chondroid matrix. Tissue heterogeneity and irregularities of the surface were observed in two cases.

Conclusions: Clinical improvement and hyaline-like appearance of the repair tissue indicate that membrane-seeded autologous chondrocyte implantation is an effective technique for the treatment of cartilage lesions.

Autogenous cancellous bone is the most effective material in stimulating osteogenic response and the standard graft augmentation for patients with nonunions or bone defects. However it may not be available in sufficient quantity and bone harvesting may give rise to morbidity. Allograft does not have the osteogenic potential of autogenous bone and may be complicated by immunological reaction and transmission of infections.

The search of bone substitutes has led to study several growth factors capable of inducing bone formation. Bone morphogenetic proteins (BMPs) have been shown to have a central role in endochondral and intramembranous bone formation and are thought to promote normal bone healing process. Recent studies demonstrated that platelet-rich plasma (PRP) provides several growth factors and stimulates osteogenesis.

The aim of the study was the evaluation of rhBMP-7 (rhOP-1) and PRP effects on the different cells detected at the site of nonunion, such as osteoblast-like cells, fibroblast-like cells and mesenchymal cells.

During the surgical treatment of seven nonunion cases, cancellous bone and nonunion tissue were harvested. Osteoblast-like cells and fibroblast-like cells were isolated and characterized. Mesenchymal cells were obtained from bone marrow samples of the same patients. Each cell type was incubated with rhBMP-7 and PRP at different concentrations. Proliferation rate and alkaline phosphatase (ALP) activity were assessed at 3, 7, 15, and 30 days. cytochemical and immunocytochemical analysis were performed at 15 and 30 days.

Proliferation rate was higher in osteoblast-like cells and mesenchymal cells than in fibroblast-like cells. Growth factors induced mesenchymal cells to express osteoblast phenotype markers.

The results show that fibroblast-like cells at the site of nonunion are responsive to growth factors stimulation, though their low osteoblastic differentiation rate, even at highest concentration of growth factors. These data suggest that the use of growth factors in nonunion treatment should be combined with autologous cancellous bone and/or bone marrow graft, sources of target cells, in order to enhance osteogenic response.

Bone marrow would represent a useful source of cells for skeletal tissue engineering. Marrow mesenchymal stem cells (MSC) can generate cartilage, bone and fat. The differentiation of this multipotent population into fibroblast, chondrocytes or osteoblasts can be inducted in vitro by the addiction of growth factor like bFGF, TGFA7, BMP-2.

In order to evaluate the possibility of inducing cell differentiation by cell-matrix interaction, we studied the in vitro behaviour of human MSC cultured on various scaffolds.

Bone marrow was obtained during surgery for pelvic fractures or hip arthroplasty. MSC were isolated by cell sorting (CD45/glycophorin A micromagnetic beads), expanded and characterised by FACSCalibur flow cytometry system (CD3, CD34, CD14, CD45, CD90 and CD105). Then cells were grown for 30 days on different scaffolds: type I and type II collagen, type I collagen + hydroxyapatite. Histochemical (alcian blue, safranin O, ALP and von Kossa stains), immunohistochemical (type I e II collagen, chondroitin sulphate, osteonectin), histomorphometric (area %) and spectrophotometric (cell proliferation, PG synthesis, ALP activity) analyses were performed after 15 and 30 days of culture.

Among the scaffolds tested in the present study, we observed a great variability in terms of MSC adhesion and proliferation. MSC grown on type II collagen differentiated into cells expressing chondrocytes markers (S100, collagen II, chondroitin-S). MSC grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells.

These data evidenced that MSC-matrix interaction can influence phenotype expression, cell adhesion and growth rate.