Our knowledge of primary bone marrow edema (BME) of the knee is still limited. A major contributing factor is that it shares several radiological findings with a number of vascular, traumatic, and inflammatory conditions having different histopathological features and etiologies.

BME can be primary or secondary. The most commonly associated conditions are osteonecrosis, osteochondritis dissecans, complex regional pain syndrome, mechanical strain such as bone contusion/bruising, micro-fracture, stress fracture, osteoarthritis, and tumor. The etiology and pathogenesis of primary BME are unclear. Conservative treatment includes analgesics, non-steroidal anti-inflammatory drugs, weight-bearing limitations, physiotherapy, pulsed electromagnetic fields, prostacyclin, and bisphosphonates. Surgical treatment, with simple perforation, fragment stabilization, combined scraping and perforation, and eventually osteochondral or chondrocyte transplant, is reserved for the late stages. This retrospective study of a cohort of patients with primary BME of the knee was undertaken to describe their clinical and demographic characteristics, identify possible risk factors, and assess treatment outcomes.

We reviewed the records of 48 patients with primary BME of the knee diagnosed on MRI by two radiologists and two orthopedists. History, medications, pain type, leisure activities, smoking habits, allergies, and environmental factors were examined. Analysis of patients’ characteristics highlighted that slightly overweight middle-aged female smokers with a sedentary lifestyle are the typical patients with primary BME of the knee. In all patients, the chief symptom was intractable day and night pain (mean value, 8.5/10 on the numerical rating scale) with active as well as passive movement, regardless of BME extent. Half of the patients suffered from thyroid disorders; indeed, the probability of having a thyroid disorder was higher in our patients than in two unselected groups of patients, one referred to our orthopedic center (odds ratio, 18.5) and another suffering from no knee conditions (odds ratio, 9.8). Before pain onset, 56.3% of our cohort had experienced a stressful event (mourning, dismissal from work, concern related to the COVID-19 pandemic). After conservative treatment, despite the clinical improvement and edema resolution on MRI, 93.8% of patients described two new symptoms: a burning sensation in the region of the former edema and a reduced ipsilateral patellar reflex. These data suggest that even though the primary BME did resolve on MRI, the knee did not achieve full healing.

Introduction and Objective

The geometry of the proximal tibia and distal femur is intimately linked with the biomechanics of the knee and it is to be considered in total knee arthroplasty (TKA) component positioning. The aim of the present study was to evaluate the proximal tibial torsion in relation to the flexion-extension axis of the knee in healthy and pathological cohort affected by knee osteoarthritis (OA).

The objective of this study was to evaluate the safety and the effect of platelet-rich plasma (PRP) intra-articular injections obtained from blood donors (homologous PRP) on elderly patients with early or moderate knee osteoarthritis (OA) who are not candidates for autologous PRP treatment.

A total of 60 symptomatic patients, aged 65–86 years, affected by hematologic disorders and early or moderate knee OA, were treated with 5 ml of homologous PRP intraarticular injections every 14 days for a total of three injections. Clinical evaluations before the treatment, and after 2 and 6 months were performed by International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS) and Equal Visual Analogue Scale (EQ VAS) scores. Adverse events and patient satisfaction were recorded.

No severe complications were noted during the treatment and the follow-up period. A statistically significant improvement from basal evaluation to the 2-month follow-up visit was observed, whereas a statistically significant worsening from the 2-month to the 6-month follow-up visit was showed. The overall worst results were observed in patients aged 80 years or over and in those affected by minor bone attrition. It was found that 90% of patients were satisfied at the 6-month evaluation.

Homologous PRP has an excellent safety profile but offers only a short-term clinical improvement in selected elderly patients with knee OA who are not candidates for autologous PRP treatment. Increasing age and developing degeneration result in a decreased potential for homologous PRP injection therapy. Further studies are needed to confirm these findings.

Nerve growth factor (NGF) is involved in several joint diseases. It participates in pain initiation, inadequate nociception and neurogenic inflammation; its concentrations are increased in synovial fluid and tissue from human and experimental arthritis. However, data about its role in normal and pathological articular cartilage are scant and conflicting. This study assesses the effects of different

NGF concentrations on cultured healthy human chondrocytes by evaluating cell proliferation, cell phenotype, and gene expression.

The 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl-2H-tetrazolium bromide (MTT) test excluded an influence on cell viability; alcian blue and S100 staining demonstrated that NGF induced de-differentiation of the chondrocyte phenotype; real-time PCR disclosed that it reduced the expression of collagen type II (COL2A1) and transforming growth factor-β (TGF-β), key factors involved in articular cartilage integrity, and stimulated upregulation of metalloproteinase (MMP)-3 and MMP-13.

These findings suggest that NGF may adversely affect differentiated chondrocytes from articular cartilage by inhibiting the expression of the collagens found in normal articular cartilage (COL2A1), while exerting a degradative effect though TGF-β downregulation and MMP-13 and MMP-3 upregulation. Further investigation is required to determine whether the gene expression pattern found in our study is associated with changes in protein expression.

Summary Statement

This experimental study showed that platelet rich fibrin matrix can improve muscle regeneration and long-term vascularization without local adverse effects.

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.

A less invasive surgical treatment of clubfoot is increasingly considered, it aims to limit extensive exposure, to improve the functional and cosmetic outcome and to lower the risk of stiffness and recurrence of the deformity. The Ponseti method consists in an original casting technique followed, only in the most resistant clubfeet, by a percutaneous Achilles tenotomy. Critical decision is the selection of the clubfeet which needs tenotomy. Purpose of this study was to determine if ultrasound assessment of clubfoot may be helpful in making surgical decision.

MATERIAL AND Methods: 98 newborns with 122 congenital clubfeet were treated by the Ponseti casting technique from mid-2000 to June 2006. According to Manes classification, there were 20 mild, 47 moderate and 55 severe clubfeet. After 3 to 8 weeks of casting, clubfeet candidate to surgery underwent sonographic assessment according to the original technique previously published by the authors. On the sagittal posterior plane the R.O.M. of the ankle and subtalar joints was stated both in neutral position and under manipulation. No surgery was performed in clubfeet with normal sonographic dorsiflexion, percutaneous tenotomy was done in clubfeet with mild limited sonographic dorsiflexion and more extensive posterior release (tendon Z-lengthening and posterior cut of ankle and subtalar joint) was performed in clubfeet with most evident sonographic persistent equinus and anterior dislodgment of the talus in the ankle mortise. The R.O.M. was checked again by ultrasound at the end of treatment. According to Ponseti method a Denis Browne bar, with clubfoot 60° externally rotated, was worn full time until the walking age.

Results: 35/122 clubfeet (28,6%) were treated conservatively (all the 20 mild and 15/47 of moderate deformities), 87/122 (71,4%) surgically (32/47 of moderate deformities and all the 55 severe deformities). On the basis of the dynamic ultrasound evaluation 38 clubfeet underwent simple tenotomy and 49 ones underwent extensive posterior release. At the end of the casting normal dorsiflexion was documented by ultrasound in 72 (82,7%) of the operated feet.

Conclusions: The need of surgery in the Ponseti casting technique shows a great variability in Literature. These controversial data are probably due not only to the different confidence in the Ponseti method, but also to the different criteria used in evaluating the correction obtained by casting. Ultrasound assessment of the deformity gives objective qualitative and quantitative information about the restoration of the physiological dorsiflexion and articular biomechanics. On the basis of this simple, non invasive and widely available procedure the surgeon can evaluate the effectiveness of the serial casting and may be able to establish and graduate the need of corrective surgery.

Forty patients with subcutaneous rupture of the Achilles tendon were enrolled in a prospective study and randomised to two groups: group A treated with open surgery with Kessler-type suture, and group B treated by percutaneous tenorraphy (Tenolig®) under ultrasound control. The follow-up included an objective and a subjective (SF-12) clinical evaluation at 4, 12 and 24 months, ultrasonography at the same time points, and isokinetic muscle performance tests at 12 months. There were no significant differences between the two groups at clinical and ultrasound evaluation except for a greater ankle circumference in group B (p< 0.01) at 12 months; peak torque and total work isokinetic tests did not differ significantly in the two groups nor between involved and uninvolved side. At 24 months data show similar clinical and ultrasonographic results with both techniques, leading us to prefer percutaneus tenorraphy under ultrasound control owing to the attendant advantages of local anaesthesia in day surgery, decreased risk of skin complications, reduced surgical time, faster functional recovery, and greater patient compliance.

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.

Introduction: In temperate places pyomyositis is very uncommon in children and adolescents. West European and North American Literature about is relatively poor (104 articles from 1998 to nowadays and most of these papers are case-rep orts). S. Aureus is the etiologic agent in 90% of cases. Muscle of the thighs and hips are the most frequent localisation and severe complications are observed in 10% of cases.

Material and Methods: We reviewed all patients affected by primary pyomyositis and admitted in our Department from 1995 to nowadays. Age, sex, history and clinical findings, general and local risk factors were reported. X-rays, Ultrasonography, MRI, Scintigraphy, Haematological investigation and culture were considered in order to state the imaging and laboratory findings of the disease.

Result: Pyomyositis was diagnosed in three males, aged 11, 13 and 16. There was no evidence of medical or familiar risk factors. Soleus, Otturatorius and Psoas muscles were respectively involved. In all cases fever, local signs of inflammation and pain, neutrophylic leukocytosis, increased ESR and CRP levels were present. Blood culture was positive in one case. Standard X-Rays were normal in all cases. Ultrasound scan, RMI and Scintigraphy were positive but non-specific in all cases. 2 cases underwent to surgical drainage and a 3 weeks antibiotic therapy; 1 case resolved performing only medical treatment (5 weeks of antibiotic). All patients showed complete clinical, haematological, and RMI recovered without reliquates.

Conclusions: Pyomyositis is uncommon in temperate climate and it may be easy misdiagnosed at the onset. In our patients the mean time from onset of symptoms to diagnosis was 10 days. History and clinical examination might be evocative, but there are no pathognomonic haematological or radiological findings so that diagnosis is often the result of extensive and wide considerations. Pyomyositis should be suspected in any unclear septic condition of the musculoskeletal apparatus. High index of suspicion, prompt diagnosis, and early treatment can prevent complications and allow the recovery of this potentially life-threatening disease.

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.

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 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.

Renal failure in children is associated with a wide range of musculoskeletal disorders such as osteonecrosis, stress fractures, brown tumours, epiphysiolysis, joint infections and angular deformities. In this paper the authors report their experience concerning the surgical treatment of the angular deformities of the lower limbs in renal osteodystrophy (RO).

Between 1995 to 2003, 10 children (five girls and five boys) with RO underwent surgical correction of angular deformities of the lower limbs. Of these, seven had femoral osteotomies because of knee deformities (three genu valgum, four genu varum) and three had osteotomies because of tibial angular deformity. The average age at surgery was 5 years (min. 2 years, max. 12 years). Different types of osteosynthesis were used (staples and cast, Ortho-fix and Ilizarov frames) according to the age of the child and the degree and the site of the angular deformities.

All osteotomies healed without complications and the surgical correction was considered appropriate at the end of treatment. At an average follow-up of 4.5 years there was no significant relapse and no need for second surgery.

Simple osteosynthesis (staples and cast) was most appropriate in the youngest children and in mildest deformities (particularly at the distal tibial metaphysis). External devices were more suitable in the oldest children and for genu valgum/varum deformities. To optimise the time of consolidation close collaboration with the paediatricians is required in order to perform surgery under the best metabolic conditions (elevation of the serum alkaline phosphatase concentration above 500/l is a good marker of bone metabolic healthy).

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.