Phenotype and Viability of MLO-Y4 Cells Is Maintained by TGFß3 in a Serum-Dependent Manner within a 3D-Co-Culture with MG-63 Cells.

The osteocyte network inside the bone matrix is of functional importance and osteocyte cell death is a characteristic feature of pathological bone diseases. Osteocytes have emerged as key regulators of bone tissue maintenance, yet maintaining their phenotype during in vitro culture remains challenging. A 3D co-culture system for osteocytes with osteoblasts was recently presented, enabling the determination of more physiological effects of growth factors on cells in vitro. MLO-Y4 cells were embedded within a type I collagen gel and cultured in the presence of surface MG-63 cells. Co-culture was performed in the presence or absence of TGFß3. Gene expression by quantitative PCR, protein expression by fluorescent immunohistochemistry and cell viability tests were performed. The 3D co-culture induced cell differentiation of MG-63 cells seen by increased type I collagen and osteocalcin mRNA expression. TGFβ3 maintained osteocyte differentiation of MLO-Y4 cells during co-culture as determined by stable E11 and osteocalcin mRNA expression till day 4. Interestingly, most of the effects of TGFß3 on co-cultured cells were serum-dependent. Also, TGFß3 reduced cell death of 3D co-cultured MLO-Y4 cells in a serum-dependent manner. This study shows that 3D co-culture upregulates differentiation of MG-63 cells to a more mature osteoblast-like phenotype; while the addition of TGFß3 maintained the characteristic MLO-Y4 osteocyte-like phenotype and viability in a serum-dependent manner.

Fibroblast growth factor 2 and transforming growth factor ß1 induce precocious maturation of articular cartilage.

OBJECTIVE: We have discovered that a combination of fibroblast growth factor 2 and transforming growth factor ß1 induce profound morphologic changes in immature articular cartilage. The purpose of this study was to test the hypothesis that these changes represent accelerated postnatal maturation. METHODS: Histochemical and biochemical assays were used to confirm the nature of the morphologic changes that accompany growth factor stimulation of immature bovine articular cartilage explants in serum-free culture medium. Growth factor-induced apoptosis, cellular proliferation, and changes in the collagen network were also quantitatively analyzed. RESULTS: Growth factor stimulation resulted in rapid resorption from the basal aspect of immature cartilage explants that was simultaneously opposed by cellular proliferation from the apical aspect driven from a pool of chondroprogenitor cells we have previously described. Maturation-dependent changes in tissue stiffness, collagen crosslinking, and collagen fibril architecture as well as differentiation of the extracellular matrix into distinct pericellular, territorial, and interterritorial domains were all present in growth factor-stimulated cartilage samples and absent in control samples. CONCLUSION: Our data demonstrate that it is possible to significantly enhance the maturation of cartilage tissue using specific growth factor stimulation. This may have applications in transplantation therapy or in the treatment of diseased cartilage, through phenotype modulation of osteoarthritic chondrocytes in order to stimulate growth and maturation of cartilage repair tissue.

Morphological changes in disc herniation in the lower cervical spine: an ultrastructural study.

INTRODUCTION: The basis of disc degeneration is still unknown, but is believed to be a cell-mediated process. Apoptosis might play a major role in degenerative disc disease (DDD). The aim of this study was to correlate the viability of disc cells with the radiological degeneration grades (rDG) in disc herniation. MATERIALS AND METHODS: Forty anterior IVD's (C4-C7) from 39 patients with DDD were studied histologically and ultrastructurally to quantify healthy, "balloon", chondroptotic, apoptotic and necrotic cells. Patients were classified to their rDG, as having either prolapse (P: DGII + III) and/or osteochondrosis (O: DGIV + V). Similar studies were undertaken on eight control discs. RESULTS: Cell death by necrosis (mean 35%) was common but differed not significantly in both groups. All patients with a disc prolapse DGII + III revealed balloon cells (iAF: mean 32%). All appeared alive and sometimes were hypertrophic. However, significantly less balloon cells were found in the O-Group. Control samples revealed no evidence of "balloon" cells in DGII and only a minor rate in DGIII. CONCLUSION: According to the different rDG, quantitative changes were obvious in healthy and "balloon" cells, but not for cell death. At the moment it can only be hypothesized if "balloon" cells are part of a repair strategy and/or cause of disc herniation.

Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap.

Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.

Bone Morphogenetic Protein-9 Is a Potent Chondrogenic and Morphogenic Factor for Articular Cartilage Chondroprogenitors.

Articular cartilage contains a subpopulation of tissue-specific progenitors that are an ideal cell type for cell therapies and generating neocartilage for tissue engineering applications. However, it is unclear whether the standard chondrogenic medium using transforming growth factor beta (TGFß) isoforms is optimal to differentiate these cells. We therefore used pellet culture to screen progenitors from immature bovine articular cartilage with a number of chondrogenic factors and discovered that bone morphogenetic protein-9 (BMP9) precociously induces their differentiation. This difference was apparent with toluidine blue staining and confirmed by biochemical and transcriptional analyses with BMP9-treated progenitors exhibiting 11-fold and 5-fold greater aggrecan and collagen type II (COL2A1) gene expression than TGFß1-treated progenitors. Quantitative gene expression analysis over 14 days highlighted the rapid and phased nature of BMP9-induced chondrogenesis with sequential activation of aggrecan then collagen type II, and negligible collagen type X gene expression. The extracellular matrix of TGFß1-treated progenitors analyzed using atomic force microscopy was fibrillar and stiff whist BMP9-induced matrix of cells more compliant and correspondingly less fibrillar. Polarized light microscopy revealed an annular pattern of collagen fibril deposition typified by TGFß1-treated pellets, whereas BMP9-treated pellets displayed a birefringence pattern that was more anisotropic. Remarkably, differentiated immature chondrocytes incubated as high-density cultures in vitro with BMP9 generated a pronounced anisotropic organization of collagen fibrils indistinguishable from mature adult articular cartilage, with cells in deeper zones arranged in columnar manner. This contrasted with cells grown with TGFß1, where a concentric pattern of collagen fibrils was visualized within tissue pellets. In summary, BMP9 is a potent chondrogenic factor for articular cartilage progenitors and is also capable of inducing morphogenesis of adult-like cartilage, a highly desirable attribute for in vitro tissue-engineered cartilage.

Distinct mesenchymal progenitor cell subsets in the adult human synovium.

OBJECTIVE: To analyse the heterogeneity at the single-cell level of human mesenchymal progenitor cells from SM. METHODS: Cell populations were enzymatically released from the knee joint synovium of adult human individuals. Single cell-derived clonal populations were obtained by limiting dilution and serially passaged to determine growth rates. Phenotypic analysis was carried out by flow cytometry. Replicative senescence was assessed by the senescence-associated beta-galactosidase staining. Telomere lengths were determined semiquantitatively by Southern blotting. Telomerase activity was measured using a real-time quantitative telomerase repeat amplification procedure. Culture-expanded clonal populations were subjected to in vitro differentiation assays to investigate their mesenchymal multipotency. RESULTS: The 50 clones analysed displayed wide variations in the proliferation rates, even within the same donor sample. The time taken to reach 20 population doublings ranged from 44 to 130 days. The phenotype of the clones tested was compatible with that of mesenchymal stem cells. Mean telomere lengths ranged from 5.2 to 10.9 kb with positive linear trend with telomerase activity, but no correlation with proliferative rates or cell senescence. All clones tested were capable of chondrogenic and osteogenic differentiation, though with large variability in potency. In contrast, only 30% of the clones were adipogenic. CONCLUSIONS: We report for the first time the co-existence, within the synovium, of progenitor cell subsets with distinct mesenchymal differentiation potency. Our findings further emphasize the need for strategies to purify cell populations with the clinically desired tissue formation potentials.

Notch receptor and Notch ligand expression in developing avian cartilage.

The development of limb cartilage involves complex signalling pathways allowing the formation of distinct segments of cartilage that are maintained in the fully developed joint. In this study, we investigated the Notch signalling pathway and its role in cartilage development. The differential distribution of the Notch signalling family of receptors and their corresponding ligands in developing avian (gallus gallus) cartilage revealed expression of Notch 1, Delta 1, Jagged 1 and Jagged 2 in all limb mesenchyme cells at the early stages of cartilage anlagen development, which were subsequently restricted to the developing cartilage element. Expression of both Notch 1 and Jagged 1 became increasingly restricted to the surface cartilage once joint cavity formation had occurred. Delta 1 and Jagged 1 were restricted to a layer of cells underneath the surface cartilage and were also observed in the hypertrophic chondrocytes, where Notch 1 expression was evident in stage 40-44 limbs. Notch 2, Notch 3 and Notch 4 were not evident in early stage limbs but were present after cavitation, although expression was lost in late stage limbs (stage 40-44). We also demonstrated that inhibition of the Notch pathway leads to altered Notch receptor expression, disrupting cartilage differentiation. From these data it is clear that Notch signalling is a necessary and critical factor in regulating cell fate decisions allowing controlled chondrogenesis, elongation and subsequent maintenance of limb cartilage.

In situ detection of cell death in articular cartilage.

Necrosis and apoptosis have been demonstrated in articular cartilage in response to trauma and disease. However, cell death in articular cartilage may also be thought of as a scale of cell death culminating in secondary necrosis with the failure to remove apoptotic cells from the tissue. The in situ detection of cell death is an important technique in studying articular cartilage as it most closely resembles the in vivo situation. The methods described here involve the use of light microscopy and electron microscopy in conjunction with fluorescent and biochemical methods to correctly ascertain the type of cell death that has occurred.

Characterisation of a divergent progenitor cell sub-populations in human osteoarthritic cartilage: the role of telomere erosion and replicative senescence.

In recent years it has become increasingly clear that articular cartilage harbours a viable pool of progenitor cells and interest has focussed on their role during development and disease. Analysis of progenitor numbers using fluorescence-activated sorting techniques has resulted in wide-ranging estimates, which may be the result of context-dependent expression of cell surface markers. We have used a colony-forming assay to reliably determine chondroprogenitor numbers in normal and osteoarthritic cartilage where we observed a 2-fold increase in diseased tissue (P < 0.0001). Intriguingly, cell kinetic analysis of clonal isolates derived from single and multiple donors of osteoarthritic cartilage revealed the presence of a divergent progenitor subpopulation characterised by an early senescent phenotype. Divergent sub-populations displayed increased senescence-associated ß-galactosidase activity, lower average telomere lengths but retained the capacity to undergo multi-lineage differentiation. Osteoarthritis is an age-related disease and cellular senescence is predicted to be a significant component of the pathological process. This study shows that although early senescence is an inherent property of a subset of activated progenitors, there is also a pool of progenitors with extended viability and regenerative potential residing within osteoarthritic cartilage.

Asymmetrical seeding of MSCs into fibrin-poly(ester-urethane) scaffolds and its effect on mechanically induced chondrogenesis.

Mesenchymal stem cells (MSCs) are currently being investigated as candidate cells for regenerative medicine approaches for the repair of damaged articular cartilage. For these cells to be used clinically, it is important to understand how they will react to the complex loading environment of a joint in vivo. In addition to investigating alternative cell sources, it is also important for the structure of tissue-engineered constructs and the organization of cells within them to be developed and, if possible, improved. A custom built bioreactor was used to expose human MSCs to a combination of shear and compression loading. The MSCs were either evenly distributed throughout fibrin-poly(ester-urethane) scaffolds or asymmetrically seeded with a small proportion seeded on the surface of the scaffold. The effect of cell distribution on the production and deposition of cartilage-like matrix in response to mechanical load mimicking in vivo joint loading was then investigated. The results show that asymmetrically seeding the scaffold led to markedly improved tissue development based on histologically detectable matrix deposition. Consideration of cell location, therefore, is an important aspect in the development of regenerative medicine approaches for cartilage repair. This is particularly relevant when considering the natural biomechanical environment of the joint in vivo and patient rehabilitation protocols. Copyright © 2016 John Wiley & Sons, Ltd.

Tissue-Engineered Solutions in Plastic and Reconstructive Surgery: Principles and Practice.

Recent advances in microsurgery, imaging, and transplantation have led to significant refinements in autologous reconstructive options; however, the morbidity of donor sites remains. This would be eliminated by successful clinical translation of tissue-engineered solutions into surgical practice. Plastic surgeons are uniquely placed to be intrinsically involved in the research and development of laboratory engineered tissues and their subsequent use. In this article, we present an overview of the field of tissue engineering, with the practicing plastic surgeon in mind. The Medical Research Council states that regenerative medicine and tissue engineering "holds the promise of revolutionizing patient care in the twenty-first century." The UK government highlighted regenerative medicine as one of the key eight great technologies in their industrial strategy worthy of significant investment. The long-term aim of successful biomanufacture to repair composite defects depends on interdisciplinary collaboration between cell biologists, material scientists, engineers, and associated medical specialties; however currently, there is a current lack of coordination in the field as a whole. Barriers to translation are deep rooted at the basic science level, manifested by a lack of consensus on the ideal cell source, scaffold, molecular cues, and environment and manufacturing strategy. There is also insufficient understanding of the long-term safety and durability of tissue-engineered constructs. This review aims to highlight that individualized approaches to the field are not adequate, and research collaboratives will be essential to bring together differing areas of expertise to expedite future clinical translation. The use of tissue engineering in reconstructive surgery would result in a paradigm shift but it is important to maintain realistic expectations. It is generally accepted that it takes 20-30 years from the start of basic science research to clinical utility, demonstrated by contemporary treatments such as bone marrow transplantation. Although great advances have been made in the tissue engineering field, we highlight the barriers that need to be overcome before we see the routine use of tissue-engineered solutions.

Mechanical regulation of secondary chondrogenesis.

The development of the skull is characterised by its dependence upon epigenetic influences. One of the most important of these is secondary chondrogenesis, which occurs following ossification within certain membrane bone periostea, as a result of biomechanical articulation. We have studied the genesis, character and function of the secondary chondrocytes of the quadratojugal of the chick between embryonic days 11 and 14. Analysis of gene expression revealed that secondary chondrocytes formed coincident with Sox9 upregulation from a precursor population expressing Cbfa1/Runx2: a reversal of the normal sequence. Such secondary chondrocytes rapidly acquired a phenotype that is a compound of prehypertrophic and hypertrophic chondrocytes, exited from the cell cycle and upregulated Ihh. Pulse and pulse/chase experiments with BrdU confirmed the germinal region as the highly proliferative source of the secondary chondrocytes, which formed by division of chondrocyte-committed precursors. By blocking Hh signalling in explant cultures we show that the enhanced proliferation of the germinal region surrounding the secondary chondrocytes derives from this Ihh source. Additionally, in vitro studies on membrane bone periosteal cells (nongerminal region) demonstrated that these cells can also respond to Ihh, and do so both by enhanced proliferation and precocious osteogenesis. Despite the pro-osteogenic effects of Ihh on periosteal cell differentiation, mechanical articulation of the quadratojugal/quadrate joint in explant culture revealed a negative role for articulation in the regulation of osteocalcin by germinal region descendants. Thus, the mechanical stimulus that is the spur to secondary chondrocyte formation appears able to override the osteogenic influence of Ihh on the periosteum, but does not interfere with the cell cycle-promoting component of Hh signalling.

Combining regenerative medicine strategies to provide durable reconstructive options: auricular cartilage tissue engineering.

Recent advances in regenerative medicine place us in a unique position to improve the quality of engineered tissue. We use auricular cartilage as an exemplar to illustrate how the use of tissue-specific adult stem cells, assembly through additive manufacturing and improved understanding of postnatal tissue maturation will allow us to more accurately replicate native tissue anisotropy. This review highlights the limitations of autologous auricular reconstruction, including donor site morbidity, technical considerations and long-term complications. Current tissue-engineered auricular constructs implanted into immune-competent animal models have been observed to undergo inflammation, fibrosis, foreign body reaction, calcification and degradation. Combining biomimetic regenerative medicine strategies will allow us to improve tissue-engineered auricular cartilage with respect to biochemical composition and functionality, as well as microstructural organization and overall shape. Creating functional and durable tissue has the potential to shift the paradigm in reconstructive surgery by obviating the need for donor sites.

Evaluation of articular cartilage progenitor cells for the repair of articular defects in an equine model.

BACKGROUND: We sought to determine the effectiveness of chondroprogenitor cells derived from autologous and allogenic articular cartilage for the repair of cartilage defects in an equine model. METHODS: Cartilage defects (15 mm) were created on the medial trochlear ridge of the femur. The following experimental treatments were compared with empty-defect controls: fibrin only, autologous chondroprogenitor cells plus fibrin, and allogenic chondroprogenitor cells plus fibrin (n = 4 or 12 per treatment). Horses underwent strenuous exercise throughout the twelve-month study, and evaluations included lameness (pain) and arthroscopic, radiographic, gross, histologic, and immunohistochemical analyses. RESULTS: Arthroscopy and microscopy indicated that defects in the autologous cell group had significantly better repair tissue compared with defects in the fibrin-only and control groups. Repair tissue quality in the allogenic cell group was not superior to that in the fibrin-only group with the exception of the percentage of type-II collagen, which was greater. Radiographic changes in the allogenic cell group were poorer on average than those in the autologous cell group. Autologous cells significantly reduced central osteophyte formation compared with fibrin alone. CONCLUSIONS: On the basis of the arthroscopic, radiographic, and histologic scores, autologous cells in fibrin yielded better results than the other treatments; allogenic cells cannot be recommended at this time.

Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

Articular cartilage progenitor cells (ACPCs) represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs). This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs) are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2). hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-ß1) concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase). To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs represent a potential alterative cell source for cartilage tissue engineering applications.

The chondrocyte.

The chondrocyte is the resident cell of cartilage that is a prominent tissue in the embryo acting as a template for the development of skeletal elements. In the adult, the distribution of permanent cartilage is much more restricted and is necessary for mechanical support, growth and movement. The cell is isolated within a voluminous extracellular matrix (ECM) that is neither vascularised nor innervated. As a result, nutrient/waste exchange occurs through diffusion and, consequently, under normal and pathological conditions, the cell is unique in its ability to exist in a low oxygen tension environment. Partly as a result of these properties, the tissue has a low reparative potential that, in the case of articular cartilage, predisposes the tissue to degenerative conditions such as arthritis that is a significant clinical problem. Cellfacts. Cytoplasmically isolated. High matrix/cell volume ratio. Do not divide after skeletal maturity unless during pathology. Major contributor to growth of the body. Most energy requirements obtained through glycolysis.

Articular Chondroprogenitor Cells Maintain Chondrogenic Potential but Fail to Form a Functional Matrix When Implanted Into Muscles of SCID Mice.

OBJECTIVE: Articular cartilage is a complex tissue comprising phenotypically distinct zones. Research has identified the presence of a progenitor cell population in the surface zone of immature articular cartilage. The aim of the present study was to determine the in vivo plasticity of articular cartilage progenitor. DESIGN: Chondropogenitor cells were isolated from bovine metacarpalphalangeal joints by differential adhesion to fibronectin. Cells were labeled with PKH26 and injected into the thigh muscle of severe-combined immunodeficient (SCID) mice. After 2 weeks, the muscles were dissected and cryosectioned. Sections were stained with safranin O and labeled for sox9 and collagen type II. Polymerase chain reaction analysis was carried out to determine plasticity for a number of tissue-specific markers. Full-depth chondrocytes acted as a control. RESULTS: Fluorescent PKH26 labeled cells were detected after 2 weeks in all samples analyzed. A cartilage pellet was present after injection of freshly isolated chondrocytes. After injection with clonal and enriched populations of chondroprogenitors, no distinct pellet was detected, but diffuse cartilage nodules were found with regions of safranin O staining and Sox9. Low levels of collagen type II were also detected. Polymerase chain reaction analysis identified the presence of the endothelial cell marker PECAM-1 in one clonal cell line, demonstrating phenotypic plasticity into the phenotype of the surrounding host tissues. CONCLUSIONS: The bovine articular cartilage progenitor cells were able to survive in vivo postimplantation, but failed to create a robust cartilage pellet, despite expressing sox9 and type II collagen. This suggests the cells require further signals for chondrogenic differentiation.

Evidence of a Viable Pool of Stem Cells within Human Osteoarthritic Cartilage.

OBJECTIVES: Osteoarthritis (OA) is a debilitating disease affecting more than 4 million people in the United Kingdom. Despite its prevalence, there is no successful cell-based therapy currently used to treat patients whose cartilage is deemed irrecoverable. The present study aimed to isolate stem cells from tibial plateaux cartilage obtained from patients who underwent total knee replacements for OA and investigate their stem cell characteristics. DESIGN: Clonally derived cell lines were selected using a differential adhesion assay to fibronectin and expanded in monolayer culture. Colony forming efficiencies and growth kinetics were investigated. The potential for tri-lineage differentiation into chondrogenic, osteogenic, and adipogenic phenotypes were analyzed using histological stains, immunocytochemistry, and reverse transcriptase polymerase chain reaction. RESULTS: Colony forming cells were successfully isolated from osteoarthritic cartilage and extensively expanded in monolayer culture. Colony forming efficiencies were consistently below 0.1%. Clonal cell lines were expanded beyond 40 population doublings but disparities were observed in the number of population doublings per day. Clonally derived cell lines also demonstrated in vitro multilineage potential via successful differentiation into chondrogenic, osteogenic, and adipogenic lineages. However, variation in the degree of differentiation was observed between these clonal cell lines. CONCLUSIONS: A viable pool of cells with stem cell characteristics have been identified within human osteoarthritic cartilage. Variation in the degree of differentiation suggests the possibility of further subpopulations of cells. The identification of this stem cell population highlights the reparative potential of these cells in osteoarthritic cartilage, which could be further exploited to aid the field of regenerative medicine.

Enhanced adenovirus transduction of hMSCs using 3D hydrogel cell carriers.

Hydrogels are increasingly being investigated as a means to implant cells for tissue engineering. One way to further enhance the repair response would be to combine the hydrogel cell carrier with gene transfer. Gene therapy, using adenoviral vectors, is an effective way to provide transient delivery of bioactive factors. However, current protocols require further optimization, especially if they are to be transferred into the clinic. This study opted to compare the efficiency of protocols for standard two-dimensional (2D) versus three-dimensional (3D), adenoviral-mediated, transduction of human mesenchymal stem cells. Two different multiplicities of infection were tested. After encapsulation in fibrin, alginate or agarose, cells were cultured for 28 days. Transduction in 3D showed a much higher efficiency, compared to standard 2D transduction protocols. In 3D, the amount of transgene produced was significantly higher, for every condition investigated. Furthermore, transduction in 3D does not require a cell culture step and can be conducted within the operating theatre. In conclusion, it was demonstrated that 3D transduction, using adenoviral vectors, is superior to standard transduction protocols in 2D. It therefore, might help increasing its administration in tissue engineering and clinical applications.

Chondrogenesis of human bone marrow-derived mesenchymal stem cells is modulated by complex mechanical stimulation and adenoviral-mediated overexpression of bone morphogenetic protein 2.

Currently available methods to treat articular cartilage defects still fail to demonstrate satisfactory outcomes for many patients. Functional tissue engineering using human bone marrow-derived mesenchymal stem cells (hMSCs) is a promising alternative approach for the treatment of these defects. This study strived to investigate the combined effect of complex mechanical stimulation and adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2) on hMSC chondrogenesis. hMSCs were encapsulated in a fibrin hydrogel and seeded into biodegradable polyurethane (PU) scaffolds. A novel three-dimensional transduction protocol was used to transduce cells with an adenovirus encoding for BMP-2 (Ad.BMP-2). Control cells were left untransduced. Cells were cultured for 7 or 28 days in a chondropermessive medium, which lacks any exogenous growth factors. Thereby, the in vivo situation is mimicked more precisely. hMSCs in fibrin-PU composite scaffolds were either left as free-swelling controls or mechanically stimulated using a custom-built bioreactor system that is able to generate joint-like forces. Outcome parameters measured were BMP-2 concentration within the culture medium, and biochemical and gene expression analysis. Mechanical stimulation resulted in an upregulation of chondrogenic genes. Further, glycosaminoglycan (GAG)/DNA ratios were elevated in mechanically stimulated groups. Transduction with Ad.BMP-2 led to a pronounced upregulation of the gene aggrecan and an upregulation of Sox9 message after 7 days. Furthermore, a synergistic effect in combination with mechanical stimulation on collagen 2 message was detected after 7 days. This synergistic increase was more than 8-fold if compared to the additive effect of the application of each stimulus on its own. However, BMP-2 overexpression consistently resulted in a trend toward decreased GAG/DNA ratios in both mechanical stimulated and unloaded groups.