Introduction

After aseptic loosening, dislocation of hip prosthesis is the most frequent complication in patients with hip arthroplasty, with annual cost of treatment from 70 to 100 million dollar (American estimation)

Aim of the study: The purpose of the study was to evaluate whether the implantation of a mixed type I-type II collagen scaffold associated with subchondral perforations, would improve the reparative outcomes of sub-chondral perforations by themselves, in rabbit articular cartilage lesions, over a period of 6 months.

Materials and Methods: Ten NZW rabbits were operated bilaterally on knees. Two chondral defects of 5 mm diameter were produced in each condyle in a standardized manner, without penetrating the subchondral bone. At random, one of these lesions was treated with sub-chondral perforations, and the other with subchondral perforations and apposition of the type I-type II collagen scaffold. The membrane was secured with a transosseus stitch. The overall number of lesions was forty; twenty treated defects and twenty control defects. Rabbits were killed after six months since surgery. Then histological and histomorphometrical evaluation were performed. The O’Driscoll score was used to evaluate the quality of reparative tissue.

Results: The amount of defect fill resulted significantly greater in treated lesions rather than in control lesions. The defect fill increased from 60% to 85% of the total area of the lesion. From a histological point of view, the quality of reparative tissue improved with the addition of the scaffold, as resulted by an increase in O’Driscoll Score. Particularly, hyaline-like tissue was detected in 75% of treated lesions, rather than in 15% of control lesions.

Conclusions: The addition of a mixed type I-type II collagen membrane enhances reparative effects given by subchondral perforations. Both the histological and structural quality of the reparative tissue, both the percentage area of defect fill result to be improved by this kind of membrane.

Aims: The use of vitamin K was proposed in the treatment of osteoporosis. Some experimental studies suggested that vitamin K might promote mesenchymal stem cells (MSCs) differentiation into osteoblasts progenitors and inhibit osteoclasts formation. In the present study we analysed the effects of vitamin K at different concentrations on human mesenchymal stem cells derived from fracture callus.

Methods: MSCs were obtained from the fracture’s site of three patients during surgical operation of osteosynthesis. Cells were grown on plastic plates in DMEM, 10% foetal bovine serum (FBS), 1% penicillin-streptomycin, 1% fungizone, 5mM beta-glycerophosphate and 50 microg/ml ascorbic acid. Half of the samples was incubated with vitamin D (10 nm) and K at different concentrations (1, 3, 10 microM). Proliferation rate (MTT colorimetric assay) and cell differentiation (FACSCalibur flow cytometry) were assessed at 3, 10 and 20 days. Immunocytochemical analysis (not-carboxylated osteocalcin and carboxylated osteocalcin) was also performed.

Results: MSCs stimulated with vitamin K and D expressed higher levels of osteoblastic markers than controls at 3, 10 and 20 days of colture.

Conclusions: This study confirmed the results obtained in previous in vitro experiments: vitamin K has osteoinductive properties on MSCs derived from fracture callus and it could play a role in reparative osteogenesis in vivo.

Aims: A new type I collagen membrane developed for use as a tendon graft was tested in vitro and in vivo.

Methods: The membrane (Opocrin, Italy) is obtained from type-I collagen harvested from equine Achilles tendon and is composed of collagen I fibres oriented in a single direction. It is isotropic, resorbable, hygroscopic and non immunogenic acellular membrane.

Primary human fibroblasts were seeded on collagen I membranes with aligned fibres (# 40133) with and randomly arranged fibres (# 40153). Cell proliferation was evaluated at 4, 8 and 12 days by spectrophotometry. Membrane sections were studied by immunohistochemistry and by confocal microscope on day 12 of culture.

The middle third of the patellar tendon was lesioned bilaterally in 10 adult male New Zealand White rabbits and repaired on the right side by a graft (# 40133). The contralateral tendon was left untreated and served as control. Animals were euthanized 1 or 6 months after surgery and the tendon grafts subjected to histological examination.

Results: The in vitro study demonstrated cell viability and proliferation already on day 4 from membrane seeding.

Cells were homogeneously distributed, with a more marked orientation along the main membrane axis in batch 40133 than in 40153. The in vivo study showed that cell orientation and differentiation in the scaffold with aligned fibres was satisfactory, with decreased cellularity, good integration with the surrounding tissue and crimp formation. Inflammatory reaction or excessive implant neovascularization were never observed.

Conclusion: The new type I collagen membrane exhibited a behaviour similar to other tendon or ligament scaffolds. Fibre orientation in the membrane with aligned fibres allowed to obtain a quick and well-oriented cell growth. The membrane appears to be suitable for application in tendon and ligament repair and substitution.

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.

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.

The use of autologous grafts for vertebral arthrodesis is associated with a number of complications that should be properly considered: pain at the harvesting site, increased blood loss, prolonged surgical time, and additional scar. Moreover, in many cases, the amount of autologous bone is insufficient. Novel materials, either natural or synthetic, are therefore needed to be used as bone substitutes in vertebral surgery. For this purpose, a number of synthetic materials have been developed, their characteristics varying considerably in terms of ostoinduction, osteoconduction, biomecanics, and cost.

In particular, clinical and experimental studies have highlighted the potential of demineralized bone matrix (DBM), alone or in combination with autologous grafts, and of collagenic mineralized matrix (Healos).

Aim of this study was the evaluation of the clinical value of these materials in vertebral surgery. We have analyzed a series of 60 patients who underwent vertebral arthrodesis by the addition of either DBM (30 cases) or Healos (30 cases).

Bone substitutes were used both in posterior-lateral arthrodeses (on one side, the other being treated with autologous graft as a control) and in intersomatic arthrodeses in association with titanium or carbon fiber scaffolds.

Patients were followed-up for a minimum 1-year interval, and evaluated with regard to clinical (Oswestry score, SF-36) and radiographic (static and dynamic X-rays, spiral CT, MRI) parameters. The area of arthrodesis was independently analyzed by three independent observers.

Clinical results showed the reliability of both materials as a tool for a stable arthrodesis, since they were found to be able to achieve results comparable to those obtained with autologous grafts in the control arm of the study.

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.

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.

The material most widely used in orthopaedics is hydroxyapatite (HA), anyway many differences are still present between synthetic HA and biological HA. The aim of this study was to compare adhesion, proliferation and differentiation of human osteoblast-like cells on hydroxyapatite discs with different porosity and on plastic cultures.

Human osteoblast-like cells were isolated from 4 young patients (mean age 24.5 years old), treated with collagenase and maintained in Dulbecco’s modified essential medium-10% fetal calf serum. Cells were plated on hydroxyapatite discs with 3 different porosities (35%, 35–55% e 55%) and on plastic cultures used as control. The proliferation was determined by the MTT colorimetric method, and alkaline phosphatase (ALP) activity was measured by a spettrophotometric method. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion was studied by scanning electron microscopic (SEM) analysis. Results were analysed by one-way analysis of variance (ANOVA).

Osteoblast proliferation on HA was three- to six-fold lower then on plastic. At 28 days, 2141 (± 350) cells/well grew on the most porous disks, with highly significant differences from controls. The ALP production was 2–3 fold lower on HA than on plastic. In the most porous disks, the mean ALP activity was of 2.95 (± 0.07) UI/well after 28 days, higher than in the other two groups. The type-I collagen and the osteonectin fluorescence reaction evidenced a cytoplasmic and a matrix labeling on HA at different porosities. SEM analysis showed osteoblasts with a flattened morphology and only few of them were metabolic active.

At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p< 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks.

Four cases are described of solitary spinal neurofibroma, a rare tumour of the spinal cord or nerve roots. Computerised tomography provided an accurate topographical definition of the tumour. Magnetic resonance imaging showed an increased T2-weighted signal and multiple areas of decreased T1- and T2-weighted signals centrally. The MR signals matched the histological examination which showed hyperplastic interfascicular connective tissue, pleomorphic cells, and tightly packed nerve fibres compressed by the surrounding loose connective tissue. Electron microscopy showed three types of cell: Schwann cells, fibroblast-like cells, and mast cells. The histological findings suggests that solitary spinal neurofibroma is a distinct pathological entity which could be diagnosed preoperatively from the MR images.

We obtained specimens of growth-plate cartilage from four patients with osteogenesis imperfecta. Light microscopy showed structural changes in the tissue and morphological changes in chondrocytes and matrix, particularly in the hypertrophic zone. There were changes in the process of calcification in the primary mineralisation zone of the cartilage. We also found histochemical changes in the matrix glycosaminoglycans (GAGs) in the zones where physiological mineralisation was disturbed and where the trabeculae were interrupted and poorly mineralised. In addition to the known molecular defects in collagen, changes in GAGs and non-collagenous proteins are important factors in the pathogenesis of the disease.