Brunella Grigolo

Transplantation of chondrocytes seeded on a hyaluronan derivative (hyaff-11) into cartilage defects in rabbits.

Different methods have been used to improve chondrocyte transplantation for the repair of articular cartilage defects. Several groups of biomaterials have been proposed as support for in vitro cell growth and for in vivo implantation. Here. we describe a new approach investigating the healing of rabbit cartilage by means of autologous chondrocytes seeded on a hyaluronan derivative referred to as Hyaff-11. Full thickness defects were created bilaterally in the weight-bearing surface of the medial femoral condyle of both femora of New Zealand male rabbits. The wounds were then repaired using both chondrocytes seeded on the biomaterial and biomaterial alone. Controls were similarly treated but received either no treatment or implants of the delivery substance. Histologic samples from in and around the defect sites were examined 1, 3 and 6 months after surgery and were scored from 0 to 16. Statistically significant differences in the quality of the regenerated tissue were found between the grafts carried out with biomaterial carrying chondrocyte cells compared to the biomaterial alone or controls. This study demonstrates the efficacy of this hyaluronan-based scaffold for autologous chondrocytes transplantation.

Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial (HYAff 11): molecular, immunohistochemical and ultrastructural analysis.

Association of biomaterials with autologous cells can provide a new generation of implantable devices for cartilage repair. Such scaffolds should provide a preformed three-dimensional shape and prevent cells from escaping into the articular cavity. Furthermore, these constructs should have sufficient mechanical strength to facilitate handling in a clinical setting and stimulate the uniform spreading of cells and their phenotype redifferentiation. The aim of this study was to verify the ability of HYAFF 11, a recently developed hyaluronic-acid-based biodegradable polymer, to support the growth of human chondrocytes and to maintain their original phenotype. This capability was assessed by the evaluation of collagen types I, II and aggrecan mRNA expression. Immunohistochemical analyses were also performed to evaluate collagen types I, II and proteoglycans synthesis. A field emission in lens scanning microscopy was utilized to verify the interactions between the cells and the biomaterial. Our data indicate that human chondrocytes seeded on HYAFF 11 express and produce collagen type II and aggrecan and downregulate the production of collagen type I. These results provide an in vitro demonstration for the therapeutic potential of HYAFF 11 as a delivery vehicle in a tissue-engineered approach towards the repair of articular cartilage defects.

Autologous Chondrocyte Transplantation in Osteochondral Lesions of the Ankle Joint

The aim of this study was to assess the repair of osteochondral defects of the ankle joint with hyaline cartilage. For this purpose we have been using a technique of autologous chondrocyte transplantation for osteochondral defects of the talus for the last two years. Until the method described in the paper, treatment methods proposed for the repair of cartilaginous defects have not been histologically effective in restoring the hyaline cartilage sheath, and in all cases the neoformation of cartilage was of a fibrocartilaginous nature with varying cellular characteristics. Clinical and histological results obtained using this surgical technique have confirmed its validity. Furthermore, neither subjective nor objective complications have been reported. Less pain and better articular function have also been observed. According to the AOFAS score, an improvement from an average score of 32/100 points preop. to 91/100 points at 24 months of follow up was obtained. Laboratory data have confirmed the presence of reconstructed cartilage with chondrocytes and expression of collagen II, characteristic of hyaline cartilage.

Arthroscopic Autologous Chondrocyte Implantation in Osteochondral Lesions of the Talus : Surgical Technique and Results

Background Autologous chondrocyte implantation (ACI) in the ankle was considered up to now an extremely technically demanding surgery with considerable morbidity for the patients. Hypothesis Hyalograft C scaffold allows arthroscopic ACI, thanks to a specifically designed instrumentation. Study Design Case series; Level of evidence, 4. Methods Forty-six patients with a mean age of 31.4 years (range, 20–47) underwent operation from 2001 to 2004. They had posttraumatic talar dome lesions, type II or IIA. In the first step of surgery, an ankle arthroscopy was performed, with cartilage harvest from the detached osteochondral fragment or from the margins of the lesion. Chondrocytes were cultured on a Hyalograft C scaffold. In the second step of surgery, the Hyalograft C patch was arthroscopically implanted into the lesion, with a specifically designed instrumentation. Lesions >5 mm deep were first filled with autologous cancellous bone. Patients were evaluated clinically with the American Orthopaedic Foot and Ankle Society (AOFAS) score preoperatively and at 12 and 36 months after surgery. At a mean time interval of 18 months, the first 3 patients underwent a second-look arthroscopy with cartilage harvest from the implant and histological examination. Results The mean preoperative AOFAS score was 57.2 ± 14.3. At the 12-month follow-up, the mean AOFAS score was 86.8 ± 13.4 (P < .0005), while at 36 months after surgery, the mean score was 89.5 ± 13.4 (P < .0005). Clinical results were significantly related to the age of patients and to previous operations for cartilage repair. The results of the histological examinations revealed hyaline-like cartilage regeneration. Conclusions The Hyalograft C scaffold and the specifically designed instrumentation allowed arthroscopic implantation of chondrocytes, with excellent clinical and histological results.

Osteochondral Lesions of the Knee: A New One-Step Repair Technique with Bone-Marrow-Derived Cells

Osteochondral lesions of the knee are defects of the cartilaginous surface and underlying subchondral bone, most frequently traumatic in origin1. These lesions are predominantly located on the medial femoral condyle, and associated ligamentous or meniscal pathology is reported in 40% of cases2,3 (Fig. 1). Biomechanical studies have demonstrated increased stress concentration on the rim of the osteochondral defect, which may have important implications for cartilage longevity4. Due to poor hyaline cartilage repair capability, larger osteochondral lesions of the knee are associated both with immediate significant clinical impairment and with symptoms appearing approximately one decade earlier than the degenerative cartilage changes that are associated with idiopathic osteoarthritis5. Fig. 1 Fig. 2 Fig. 1 Osteochondral lesions can affect all areas of articular cartilage, but the femoral condyle is most often involved. Fig. 2 Bone marrow is aspirated from the posterior iliac crest. Surgery is frequently needed to treat knee symptoms in patients with osteochondral lesions of the knee and to restore the cartilage on the articular surface, which lessens the risk of the development of osteoarthritis6-8. Various surgical options have been proposed for osteochondral repair6-9 but only a few have shown the ability to provide repair of the lesion site with hyaline cartilage5,10-12. Traditionally, hyaline cartilage repair has been achieved through cartilage replacement (osteoarticular transfer system [OATS; Arthrex, Naples, Florida], which is a type of mosaicplasty)13 or cartilage regeneration through autologous chondrocyte implantation6,14. Cartilage replacement procedures have the advantage of repairing cartilage defects with use of already mature autologous cartilage cells; however, donor-site pathology, discontinuity in the orientation of the cartilage plugs, and fibrocartilage in the gaps are disadvantages of these techniques13. Cartilage regeneration by autologous chondrocyte …

Surgical Treatment of Osteochondral Lesions of the Talus in Young Active Patients

C artilage is a smooth, highly specialized tissue that coats the surface of the joint. Although it is only a few millimeters thick, it has exceptional stiffness to compression and resilience and is able to distribute loads1. It is susceptible to injury and is limited in regenerative capability2. The biological function of cartilage is to permit articular movement while minimizing surface friction, to absorb loads in the weight-bearing joints, and to reduce the stress on the subchondral bone. Figs. 1-A and 1-B Histological appearance of hyaline cartilage. Fig. 1-A After staining with safranin O (×30). Fig. 1-B After staining with alcian blue (×100). Hyaline (articular) cartilage consists of 70% water; 15% collagens (primarily type II); and 15% proteoglycans (in particular, aggrecan), noncollagen proteins, lipids, and inorganic material. Chondrocytes, the only cell type in this tissue, sit within the matrix of proteoglycans and collagen, which give the cartilage its compressive and tensile properties3 (Figs. 1-A and 1-B). Osteochondral lesions of the talus are defects of the cartilaginous surface and underlying subchondral bone of the talar dome4. The etiology of osteochondral lesions of the talus can be divided into primary and secondary. Primary osteochondral lesions of the talus represent chronic diseases of the subchondral bone, most likely due to a deficiency of the vascular supply. More rarely, they are described as associated with endocrine disorders and genetic abnormalities4. Primary osteochondral lesions were formerly described as osteochondritis dissecans5. Secondary osteochondral lesions of the talus most likely occur as a result of ankle injuries (ankle sprain or fracture), chronic instability, axial defects of the lower leg, or dysbaric osteonecrosis6,7. Because joint cartilage has a poor reparative capability, osteochondral lesions of the talus rarely heal spontaneously. More frequently, patients with osteochondral lesions of …

One-Step Cartilage Repair with Bone Marrow Aspirate Concentrated Cells and Collagen Matrix in Full-Thickness Knee Cartilage Lesions Results at 2-Year Follow-up

Objective: The purpose of our study was to determine the effectiveness of cartilage repair utilizing 1-step surgery with bone marrow aspirate concentrate (BMAC) and a collagen I/III matrix (Chondro-Gide, Geistlich, Wolhusen, Switzerland). Materials and Methods: We prospectively followed up for 2 years 15 patients (mean age, 48 years) who were operated for grade IV cartilage lesions of the knee. Six of the patients had multiple chondral lesions; the average size of the lesions was 9.2 cm2. All patients underwent a mini-arthrotomy and concomitant transplantation with BMAC covered with the collagen matrix. Coexisting pathologies were treated before or during the same surgery. X-rays and MRI were collected preoperatively and at 1 and 2 years’ follow-up. Visual analog scale (VAS), International Knee Documentation Committee (IKDC), Knee injury and Osteoarthritis Outcome Score (KOOS), Lysholm, Marx, SF-36 (physical/mental), and Tegner scores were collected preoperatively and at 6, 12, and 24 months’ follow-up. Four patients gave their consent for second-look arthroscopy and 3 of them for a concomitant biopsy. Results: Patients showed significant improvement in all scores at final follow-up (P < 0.005). Patients presenting single lesions and patients with small lesions showed higher improvement. MRI showed coverage of the lesion with hyaline-like tissue in all patients in accordance with clinical results. Hyaline-like histological findings were also reported for all the specimens analyzed. No adverse reactions or postoperative complications were noted. Conclusion: This study showed that 1-step surgery with BMAC and collagen I/III matrix could be a viable technique in the treatment of grade IV knee chondral lesions.

Comparison of Platelet-Rich Plasma Formulations for Cartilage Healing An in Vitro Study

BACKGROUND Platelet-rich plasma (PRP) has been advocated as one treatment for cartilage tissue regeneration. To date, several different platelet-rich formulations have been available, but a deep knowledge of their composition and mechanism of action in a specific clinical use is needed. The aim of this study was to investigate the effect of various PRP formulations on human chondrocytes in vitro. METHODS Blood from ten human volunteers was used to prepare three formulations: (1) PRP with a relatively low concentration of platelets and very few leukocytes (P-PRP), (2) PRP with high concentrations of both platelets and leukocytes (L-PRP), and (3) platelet-poor plasma (PPP). Selected growth factors in the formulations were measured, and the in vitro effects of various concentrations were tested by exposing chondrocytes isolated from osteoarthritic cartilage of four different men and measuring cell proliferation, matrix production, and gene expression. RESULTS L-PRP contained the highest levels of growth factors and cytokines. All three formulations stimulated chondrocyte proliferation throughout the culture period evaluated; the only significant difference among the formulations was on day 7, when P-PRP induced greater cell growth compared with the other two formulations. P-PRP stimulated chondrocyte anabolism, as shown by the expression of type-II collagen and aggrecan, whereas L-PRP promoted catabolic pathways involving various cytokines. However, L-PRP induced greater expression of the hyaluronic acid synthase-2 gene and greater production of hyaluronan compared with P-PRP. CONCLUSIONS L-PRP and P-PRP induced distinct effects on human articular chondrocytes in vitro, possibly because of differences in the concentrations of platelets, leukocytes, growth factors, and other bioactive molecules. The identification of the optimal amounts and ratios of these blood components could ideally lead to a formulation more suitable for the treatment of cartilage lesions.

Molecular and Immunohistological Characterization of Human Cartilage Two Years Following Autologous Cell Transplantation

BACKGROUND There are only a few studies concerning the cellular, biochemical, and genetic processes that occur during the remodeling of graft tissue after autologous chondrocyte transplantation. The purpose of the present study was to quantify the expression of genes encoding extracellular matrix proteins and regulatory factors that are essential for cell differentiation in cartilage biopsy specimens from patients who had this treatment two years previously. METHODS Two cartilage biopsy specimens from each of four patients who had been treated with autologous chondrocyte transplantation and from two multiorgan donors were used. Real-time reverse transcriptase-polymerase chain reaction analysis was performed to evaluate the expression of types I, II, and X collagen; aggrecan; cathepsin B; and early growth response protein-1 (Egr-1) and Sry-type high-mobility-group box transcription factor-9 (Sox-9) mRNAs. Immunohistochemical analysis for matrix proteins and regulatory proteins was carried out on paraffin-embedded sections. RESULTS Type-I collagen mRNA was expressed in all of the samples evaluated. Type-II collagen was present in autologous chondrocyte transplantation samples but at lower levels than in the controls. Type-X collagen messenger was undetectable. Aggrecan mRNA was present in all of the samples at lower levels than in the controls, while cathepsin-B messenger levels were higher and Egr-1 and Sox-9 mRNAs were expressed at lower levels. The immunohistochemical analysis showed slight positivity for type-I collagen in all of the sections. Type-II collagen was found in all of the samples with positivity confined inside the cells, while the controls displayed a positivity that was diffuse in the extracellular matrix. Cathepsin B was slightly positive in all of the samples, while the controls were negative. Egr-1 protein was particularly evident in the areas negative for type-II collagen. Sox-9 was positive in all samples, with evident localization in the superficial and middle layers. CONCLUSIONS In biopsy specimens from autologous chondrocyte transplantation tissue at two years, there is evidence of the formation of new tissue, which displays varying degrees of organization with some fibrous and fibrocartilaginous features. Long-term follow-up investigations are needed to verify whether, once all of the remodeling processes are completed, the newly formed tissue will acquire the more typical features of articular cartilage.

Bipolar Fresh Osteochondral Allograft of the Ankle

Background: Severe post-traumatic ankle arthritis poses a reconstructive challenge in the young and active patient. Bipolar fresh osteochondral allograft (BFOA) may represent an intriguing alternative to arthrodesis and prosthetic replacement. The aim of this study was to describe a lateral trans-malleolar technique for BFOA, and to evaluate the results in a case series. Materials and Methods: From 2004 to 2006, 32 patients, mean age of 36.8 ± 8.4 years, affected by ankle arthritis underwent BFOA with a mean followup of 31.2 months. The graft was prepared by specifically designed jigs, including the talus and the tibia with the medial malleolus. The host surfaces were prepared by the same jigs through a lateral approach. The graft was placed and fixed with twist-off screws. Patients were evaluated clinically and radiographically at 2, 4, and 6 month after operation, and at a minimum 24 months followup. A biopsy of the grafted areas was obtained from 7 patients at 1-year followup for histological and immunohistochemical examination. Results: Preoperative AOFAS score was 33.1 ± 10.9 and postoperatively 69.5 ± 19.4 (p < 0.0005). Six failures occurred. Cartilage harvests showed hyaline-like histology with a normal collagen component but low proteoglycan presence and a disorganized structure. Samples were positive for MMP-1, MMP-13 and Capsase-3. Conclusion: The use of BFOA represents an intriguing alternative to arthrodesis or arthroplasty. We believe precise allograft sizing, stable fitting and fixation and delayed weightbearing were key factors for a successful outcome. Further research regarding the immunological behavior of transplanted cartilage is needed. Level of Evidence: IV, Retrospective Case Series