G. M. Peretti

Minced Umbilical Cord Fragments as a Source of Cells for Orthopaedic Tissue Engineering: An In Vitro Study

A promising approach for musculoskeletal repair and regeneration is mesenchymal-stem-cell- (MSC-)based tissue engineering. The aim of the study was to apply a simple protocol based on mincing the umbilical cord (UC), without removing any blood vessels or using any enzymatic digestion, to rapidly obtain an adequate number of multipotent UC-MSCs. We obtained, at passage 1 (P1), a mean value of 4, 2 × 106 cells (SD 0,4) from each UC. At immunophenotypic characterization, cells were positive for CD73, CD90, CD105, CD44, CD29, and HLA-I and negative for CD34 and HLA-class II, with a subpopulation negative for both HLA-I and HLA-II. Newborn origin and multilineage potential toward bone, fat, cartilage, and muscle was demonstrated. Telomere length was similar to that of bone-marrow (BM) MSCs from young donors. The results suggest that simply collecting UC-MSCs at P1 from minced umbilical cord fragments allows to achieve a valuable population of cells suitable for orthopaedic tissue engineering.

An in vitro tissue-engineered model for osteochondral repair

One of the main topics of regenerative medicine and tissue engineering is to address the problem of lesions involving articular cartilage. In fact, these lesions do not heal spontaneously and often lead to osteoarthritis, which causes chronic pain and worsens quality of life. Moreover, the only available treatment for osteoarthritis is symptomatic therapy and prosthetic replacement, with far from satisfactory results. A more conservative approach that restores the articular surface and function with a biologic tissue is desirable. Several strategies for regenerating articular cartilage have been proposed and applied in clinical practice but a gold standard has not yet been identified. Biphasic composites are the latest products of tissue engineering applied to articular cartilage and they seem to permit a more efficient integration of the engineered neo-tissue with the host. We present an in vitro tissue engineered model for osteochondral repair based on a composite of chondrocytes-fibrin glue gel and a calciumphosphate scaffold. This composite showed a gross integration of the two components and a cartilage-like quality of the newly formed matrix. Further studies are planned to quantify the adherence between the scaffold and the cellular fibrin glue.

Allogeneic Umbilical Cord-Derived Mesenchymal Stem Cells as a Potential Source for Cartilage and Bone Regeneration: An In Vitro Study

Umbilical cord (UC) may represent an attractive cell source for allogeneic mesenchymal stem cell (MSC) therapy. The aim of this in vitro study is to investigate the chondrogenic and osteogenic potential of UC-MSCs grown onto tridimensional scaffolds, to identify a possible clinical relevance for an allogeneic use in cartilage and bone reconstructive surgery. Chondrogenic differentiation on scaffolds was confirmed at 4 weeks by the expression of sox-9 and type II collagen; low oxygen tension improved the expression of these chondrogenic markers. A similar trend was observed in pellet culture in terms of matrix (proteoglycan) production. Osteogenic differentiation on bone-graft-substitute was also confirmed after 30 days of culture by the expression of osteocalcin and RunX-2. Cells grown in the hypertrophic medium showed at 5 weeks safranin o-positive stain and an increased CbFa1 expression, confirming the ability of these cells to undergo hypertrophy. These results suggest that the UC-MSCs isolated from minced umbilical cords may represent a valuable allogeneic cell population, which might have a potential for orthopaedic tissue engineering such as the on-demand cell delivery using chondrogenic, osteogenic, and endochondral scaffold. This study may have a clinical relevance as a future hypothetical option for allogeneic single-stage cartilage repair and bone regeneration.

Minced umbilical cord fragments as a source of cell for orthopaedic tissue engineering : an in vitro study

Objectives: Anatomic ACL reconstruction has become an accepted technique to restore functional stability to the knee. Bony landmarks have been described which aid placement of the graft in mid bundle or specific anteromedial/posterolateral bundle positions. The objective of this study was to produce objective quantitative validation of the lateral intercondylar ridge and bifurcate ridge using micro CT technology, so a to better understand these important anatomical landmarks. Methods: Cadaveric human knees were imaged using a XT 320 H LC subjects (mean length 9.3 mm; mean height 1.5 mm) with the bifurcate ridge present in 14% (mean length 4.3 mm, height 0.9 mm). A relief map of the ACL footprint was produced which showed the individual variability of the anatomical ridges. Conclusions: This study demonstrates that the bony anatomical landmarks of the ACL footprint are a variable entity in individuals. When present, they can be used as a guide for femoral tunnel placement in ACL reconstruction. However, if absent, other techniques need to be adopted such as the ‘ruler technique’ or intraoperative fluoroscopy, so as to be able to reproducibly replicate anatomic tunnel positioning.

Autologous and heterologous adipose derived stem cells seeded on oligo(polyethylene glycol) fumarate hydrogel scaffold are effective in the regeneration of critical osteochondral defects in minipig model

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...

Tissue engineering for cartilage repair: in vitro development of an osteochondral scaffold. Abstracts of the 3rd TERMIS (Tissue Engineering & Regenerative Medicine International Society) World Congress 2012. September 5-8, 2012. Vienna, Austria

Adequate cellular in-growth into biomaterials is one of the fundamental requirements in regenerative medicine. Type-I-collagen is the most commonly used material for soft tissue engineering, because it is nonimmunogenic and a highly porous network for cellular support. However, adequate cell in-growth and cell seeding has been suboptimal. Different densities of collagen scaffolds (0.3% to 0.8% (w/v)) with/without polymer knitting (poly-caprolactone (PCL)) were prepared. The structure of collagen scaffolds was characterized using scanning electronic microscopy (SEM) and HE staining. The mechanical strength of hybrid scaffolds was determined using tensile strength analysis. Cellular penetration and interconnectivity were evaluated using fluorescent bead distribution and human bladder smooth muscle cells and urothelium seeding. SEM and HE analysis showed the honeycomb structure and the hybrid scaffolds were adequately connected. The hybrid scaffolds were much stronger than collagen alone. The distribution of the beads and cells were highly dependent on the collagen density: at lower densities the beads and cells were more evenly distributed and penetrated deeper into the scaffold. The lower density collagen scaffolds showed remarkably deeper cellular penetration and by combining it with PCL knitting the tensile strength was enhanced. This study indicated that a 0.4% hybrid scaffold strengthened with knitting achieved the best cellular distribution.Human adult heart harbors a population of resident progenitor cells that can be isolated by Sca-1 antibody and expanded in culture. These cells can differentiate into cardiomyocytes and vascular cells in vitro and contribute to cardiac regeneration in vivo. However, when directly injected as single cell suspension, the survival rate and retention is really poor, less than 1% of injected cells being detectable in the hosttissue within few weeks. The present study aimed at investigating the possibility to produce scaffoldless, thick cardiac progenitor cell-derived cardiac patches by thermo-responsive technology. Human cardiac progenitors obtained from the auricles of patients were cultured as scaffoldless engineered tissues fabricated using temperature-responsive surfaces obtained by poly-N-isopropylacrylamide (PNIPAAm) surface immobilization. In the engineered tissue, progenitor cells established proper three-dimensional intercellular relationships and produced abundant extracellular matrix, while preserving their phenotype and plasticity. Cell phenotype and viability within the 3D construct were followed for 1 week, showing that no significant differentiation or apoptotic events occurred within the construct. After engineered tissues were leant on visceral pericardium, a number of cells migrated into the myocardium and in the vascular walls, where they integrated in the respective textures. The study demonstrates the suitability of such approach to deliver stem cells.Spinal cord injury and repair is one of the important focus areas in tissue regeneration. Mechanical trauma caused due to factors such as contusion, compression or involuntary stretching induce post-traumatic secondary tissue damage in many Spinal Cord Injury (SCI) patients. Therefore, there is a need for scaffolds that provide a conducive threedimensionsal (3D) environment for injured cells to attach and grow. In this study we propose to synthesize 3D polymeric scaffolds in order to study the mechanical and adhesive properties & the nature of the interactions between hyaluronan-based (HY) biomaterials and cells and tissues both in vitroandin vivo. Here we have synthesized 3D HY-based hydrogels with robust mechanical and adhesive properties and demonstrate the use of this material for neuronal-related applications such as the treatment of SCI. Cell culture and survivability studies were done with NSC-34 cells. Live/Dead assay performed on the cells revealed significant differences in the staining of live cells and showed increased viability and proliferation. The number of live cells in the HY-based hydrogels with 0.1% collagen showed higher cell numbers compared with the other hydrogels. In this study we show that Injectable HYbased hydrogels with high elasticity, comparable to the mechanical properties of nervous tissue have been used in this study to study their biocompatibility and neuroprotective properties and they show better affinity for neuronal cells.Calcium phosphates (CaP) obtained by biomineralisation in Simulated Boby Fluid have been used for decades to assess the mineralisation capability of biomaterials. Recently, they have been envisioned as potential agents to promote bone formation. In this study, we have fabricated and coated with calcium phosphate melt electrospun scaffolds whereby macropores permit adequate cell migration and nutrient transfer. We have systematically investigated the effect of coating and osteoinduction onto the response of ovine osteoblasts and we observed that the coating up-regulated alkaline phosphatase activity regardless of the in vitro culture conditions. Micro Computed Tomography revealed that only scaffolds cultured in an osteoinductive cocktail were capable of depositing mineralised matrix, and that CaP coated scaffolds were more efficient at promoting mineralisation. Theses scaffolds were subcutaneously implanted in athymic rats and this demonstrated that the osteoinduction was a pre-requisite for bone formation in this ectopic model. It showed that although the bone formation was not significantly different after 8 weeks, the CaP coated scaffolds were superior at inducing bone formation as evidenced by higher levels of mineralisation at earlier time points. This work demonstrated that CaP coating is not sufficient to induce bone formation; however the combination of osteoinduction and CaP coating resulted in earlier bone formation in an ectopic model.Introduction: Bladder regeneration using minced bladder mucosa is an alternative to costly and time-consuming conventional in vitro culturing of urothelial cells. In this method, the uroepithelium ...

A tissue engineered osteochondral composite for cartilage repair: an in vivo study

This work aimed to validate the efficacy of a tissue engineered osteochondral composite for the treatment of cartilage lesion produced in adult pigs. The osteochondral composite was manufactured by combining an osteo-compatible cylinder and a neocartilagineous tissue obtained by seeding swine articular chondrocytes into a collagen scaffold. Articular cartilage was harvested from the trochlea of six adult pigs and was enzymatically digested to isolate the chondrocytes [Deponti D.et al. 2005]. The cells were then expanded in monolayer culture in chondrogenic medium and seeded onto a collagen scaffold. The collagen scaffold was preintegrated in vitro, macroscopically and microscopically, to a an osteo-compatible cylinder. The seeded osteochondral scaffolds were left in standard culture condition for 3 weeks with the addition of growth factors. At the end of culture time the osteochondral scaffolds were surgically implanted in osteochondral lesion performed in the trochlea of the same pigs from which the cartilage was initially harvested. As control, some osteochondral lesions were treated with acellular scaffolds and others were left untreated. After 3 months, the repair tissue of the three experimental groups was macroscopically analyzed and processed for histological and biochemical analysis. The hystologic ICRS II scale showed a statistically significant difference between the three experimental groups only in the parameters regarding the cell morphology and the surface/superficial assessment: the lesion treated with the unseeded osteochondral scaffolds showed higher values in chondrocytes morphology and in the superficial layer recovery, with respect to the lesions treated with the seeded scaffolds or left untreated. The biochemical analysis showed a higher DNA content in the lesion repaired with cellular scaffold and a higher GAGs/DNA ratio in the lesions with a spontaneous repair. The result of this study demonstrate that an osteochondral scaffold was able to repair an osteochondral lesion in an in vivo model of adult pigs, showing a good integration with the surrounding tissue. The quality of the repair was higher when the scaffold was not seeded with chondrocytes, but filled with cells migrated from subchondral bone. This tissue engineered osteochondral composite could represent a valuable model for further in vivo studies on the repair of chondral/osteochondral lesion.

495 BONDING OF MENISCAL TISSUE WITH CELLULAR FIBRIN GLUE: A NUDE MOUSE STUDY

C. Cournil-Henrionnet Sr1, J. Goebel1, L. Galois2, C. Huselstein3, D. Mainard2, D. Bensoussan4, P. Netter1, J. Stoltz3, P. Gillet1, A. Pinzano-Watrin1. 1UMR 7561 CNRS-Nancy Universite, Vandoeuvre les Nancy, FRANCE, 2Chirurgie Orthopédique et Traumatologique, CHU Nancy, FRANCE, 3UMR 7563 CNRS-Nancy Universite, Vandoeuvre les Nancy, FRANCE, 4Unite de Therapie Cellulaire et Tisulaire, CHU Nancy Brabois, FRANCE

P387 EFFECT OF BLOOD ON THE MORPHOLOGICAL, BIOCHEMICAL, AND BIOMECHANICAL PROPERTIES OF NEO-CARTILAGE, SYNTHESIZED BY ISOLATED CHONDROCYTES PRE-SEEDED ONTO A BIOLOGICAL SCAFFOLD

Conclusions: 3D tissue culture of expanded human mastoidderived periosteal progenitor cells in resorbable PGLA fleeces initiates bone and cartilage formation on the cellular and molecular level. The generation of different mesenchymal tissue makes 3D tissue cultured periosteal progenitor cells to promising candidates for the treatment of OA. Further investigations with periosteal progenitor cells from donors with OA were necessary for an autologous therapy strategy.