Livia Roseti

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.

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.

Standard operating procedure for the good manufacturing practice-compliant production of human bone marrow mesenchymal stem cells.

According to the European Regulation (EC 1394/2007), Mesenchymal Stem Cells expanded in culture for clinical use are considered as Advanced Therapy Medicinal Products. As a consequence, they must be produced in compliance with Good Manufacturing Practice in order to ensure safety, reproducibility, and efficacy. Here, we report a Standard Operating Procedure describing the Good Manufacturing Practice-compliant production of Bone Marrow-derived Mesenchymal Stem Cells suitable for autologous implantation in humans. This procedure can be considered as a template for the development of investigational medicinal Mesenchymal Stem Cells-based product protocols to be enclosed in the dossier required for a clinical trial approval. Possible clinical applications concern local uses in the regeneration of bone tissue in nonunion fractures or in orthopedic and maxillofacial diseases characterized by a bone loss.

Development of Human Chondrocyte–Based Medicinal Products for Autologous Cell Therapy

A cell therapy is a clinical treatment including an ex vivo cell manipulation step. Such a therapeutical option began more than forty years ago and is now a worldwide reality. Many human cell-based clinical trials have been developed in every medicine’s field mostly to cure diseases where conventional treatments are inadequate. Even though there have been few completed trials and some conflicting results on their effectiveness have been reported, the full potential of cell-based treatments remains to be explored and investigated (Park et al., 2008). Moreover, public expectation for such novel therapies, especially for treating incurable and/or rare diseases, remains high. Nowadays each tissue of the human body, including foetal and embryonic ones, can become a reliable source for cell therapy (Mason & Dunnill, 2009). Cells isolated from a specific source can be used also to cure every other tissue of the body and may be administered alone, in combination with biomaterials, scaffolds, cytokines and growth factors or can be genetically manipulated (gene therapy). Cell administrations can be local or systemic, singles or multiples. Treatments may be autologous or allogeneic (from living or cadaver donors). A cell preparation can be crucial for a treatment such as in bone marrow transplantation or otherwise it may be used as an adjuvant to improve clinical results like in regenerative medicine or to slow down the development of several chronic conditions. Cell effect after treatment can be via the ability to differentiate along several lineages or, as recently highlighted for stem cells, also via the capacity to release anti-inflammatory cytokines, growth factors and proteins, collectively known as paracrine factors, which may modulate the host microenvironment by stimulating endogenous stem cells recruitment, differentiation and angiogenesis, thus acting as real drugs (Yagi et al., 2010). Ex vivo cell manipulation protocols are different, depending on cell source, type, target, disease and Country regulations. Current European cell therapy laws classify manipulation types according to potentially associated risks. Cutting, grinding, shaping, centrifugation, soaking in antibiotic or antimicrobial solutions, sterilization, irradiation, cell separation, concentration or purification, filtering, lyophilization, freezing, cryopreservation and vitrification are considered “minimal manipulations”. On the other

Host Environment: Scaffolds and Signaling (Tissue Engineering) Articular Cartilage Regeneration: Cells, Scaffolds, and Growth Factors

Despite being a known problem for a long time, cartilage restoration is a relatively new field in orthopaedics. Multiple factors should be considered, such patient profile and the type of lesion. The situation is complicated by the lack of ability to self-repair of this tissue. For such reasons, there is no uniform approach to managing cartilage defects.Autologous chondrocyte transplantation is successfully applied to repair chondral or osteochondral defects and, lately, osteoarthritis early lesions. However, due to several disadvantages, variations of the technique and new strategies have been developed, converging in the tissue engineering approach, which associates to the cells biomimetic scaffolds that can be also treated with soluble or mechanical stimuli to enhance the regenerative potential.To overcome issues due to the loss of chondrocyte phenotype after expansion in monolayer, alternative cell populations have been investigated, such as stem cells, which present a higher stability in culture as well as other properties that make them attractive.Recently, to deal with the problems arising from the high costs of cell expansion and the need of a two-step surgery, natural growth factors combinations, like Platelet Rich Plasma and Bone Marrow Concentrate, have been investigated, with good outcomes. A drawback is the difficulty to standardize the preparations, due to patient variability.Although a variety of developments and good results, there is still a gap between research and clinical application of cartilage tissue engineering. To fill this gap, research should try to identify the optimal scaffold, fabrication technique, cell source, and signalling factor.

Media fill for validation of a good manufacturing practice-compliant cell production process

According to the European Regulation EC 1394/2007, the clinical use of Advanced Therapy Medicinal Products, such as Human Bone Marrow Mesenchymal Stem Cells expanded for the regeneration of bone tissue or Chondrocytes for Autologous Implantation, requires the development of a process in compliance with the Good Manufacturing Practices. The Media Fill test, consisting of a simulation of the expansion process by using a microbial growth medium instead of the cells, is considered one of the most effective ways to validate a cell production process. Such simulation, in fact, allows to identify any weakness in production that can lead to microbiological contamination of the final cell product as well as qualifying operators. Here, we report the critical aspects concerning the design of a Media Fill test to be used as a tool for the further validation of the sterility of a cell-based Good Manufacturing Practice-compliant production process.

Three-Dimensional Bioprinting of Cartilage by the Use of Stem Cells: A Strategy to Improve Regeneration

Cartilage lesions fail to heal spontaneously, leading to the development of chronic conditions which worsen the life quality of patients. Three-dimensional scaffold-based bioprinting holds the potential of tissue regeneration through the creation of organized, living constructs via a “layer-by-layer” deposition of small units of biomaterials and cells. This technique displays important advantages to mimic natural cartilage over traditional methods by allowing a fine control of cell distribution, and the modulation of mechanical and chemical properties. This opens up a number of new perspectives including personalized medicine through the development of complex structures (the osteochondral compartment), different types of cartilage (hyaline, fibrous), and constructs according to a specific patient’s needs. However, the choice of the ideal combination of biomaterials and cells for cartilage bioprinting is still a challenge. Stem cells may improve material mimicry ability thanks to their unique properties: the immune-privileged status and the paracrine activity. Here, we review the recent advances in cartilage three-dimensional, scaffold-based bioprinting using stem cells and identify future developments for clinical translation. Database search terms used to write this review were: “articular cartilage”, “menisci”, “3D bioprinting”, “bioinks”, “stem cells”, and “cartilage tissue engineering”.