Amad Awadallah

A quadripotential mesenchymal progenitor cell isolated from the marrow of an adult mouse.

Adult marrow contains mesenchymal progenitor cells (MPCs) that have multiple differentiation potentials. A conditionally immortalized MPC clone, BMC9, has been identified that exhibits four mesenchymal cell phenotypes: chondrocyte, adipocyte, stromal (support osteoclast formation), and osteoblast. The BMC9 clone, control brain fibroblasts and another marrow‐derived clone, BMC10, were isolated from a transgenic mouse (H‐2Kb‐tsA58) containing a gene for conditional immortality. To test for chondrogenic potential, cells were cultured in defined medium containing 10 ng/ml transforming growth factor β and 10−7 M dexamethasone in 15‐ml polypropylene tubes (“aggregate cultures”). Adipogenic potential was quantitated by flow cytometry of Nile Red–stained cells cultured for 1 and 2 weeks in medium containing isobutyl methylxanthine, indomethacin, insulin, and dexamethasone. Support of osteoclast formation was measured by quantitating multinucleated tartrate‐resistant acid phosphatase–positive cells in spleen cell cocultures of test clones (immortomouse clones and positive control ST2 cells) cultured in the presence of 10−7 M vitamin D3 and 150 mM ascorbate‐2‐phosphate. In vivo osteogenic potential was assayed by histologic examination of bone formation in subcutaneous implants, into athymic mouse hosts, of a composite of cells combined with porous calcium phosphate ceramics. The bone marrow–derived clone BMC9 has the potential to express each of the four mesenchymal characteristics tested, while brain fibroblasts, tested under identical conditions, did not exhibit any of these four mesenchymal characteristics. BMC10 cells exhibited osteogenic and chondrogenic phenotypes, but showed only minimal expression of adipocytic or osteoclast‐supportive phenotypes. Clone BMC9 is, minimally, a quadripotential MPC isolated from the marrow of an adult mouse that can differentiate into cartilage and adipose, support osteoclast formation, and form bone. The BMC9 clone is an example of an adult‐derived multipotential progenitor cell that is situated early in the mesenchymal lineage.

Tissue-Engineered Fabrication of an Osteochondral Composite Graft Using Rat Bone Marrow-Derived Mesenchymal Stem Cells

This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.

Treatment of Osteochondral Defects with Autologous Bone Marrow in a Hyaluronan-Based Delivery Vehicle

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.

Immunochemical and Mechanical Characterization of Cartilage Subtypes in Rabbit

Cartilage is categorized into three general subgroups, hyaline, elastic, and fibrocartilage, based primarily on morphologic criteria and secondarily on collagen (Types I and II) and elastin content. To more precisely define the different cartilage subtypes, rabbit cartilage isolated from joint, nose, auricle, epiglottis, and meniscus was characterized by immunohistochemical (IHC) localization of elastin and of collagen Types I, II, V, VI, and X, by biochemical analysis of total glycosaminoglycan (GAG) content, and by biomechanical indentation assay. Toluidine blue staining and safranin-O staining were used for morphological assessment of the cartilage subtypes. IHC staining of the cartilage samples showed a characteristic pattern of staining for the collagen antibodies that varied in both location and intensity. Auricular cartilage is discriminated from other subtypes by interterritorial elastin staining and no staining for Type VI collagen. Epiglottal cartilage is characterized by positive elastin staining and intense staining for Type VI collagen. The unique pattern for nasal cartilage is intense staining for Type V collagen and collagen X, whereas articular cartilage is negative for elastin (interterritorially) and only weakly positive for collagen Types V and VI. Meniscal cartilage shows the greatest intensity of staining for Type I collagen, weak staining for collagens V and VI, and no staining with antibody to collagen Type X. Matching cartilage samples were categorized by total GAG content, which showed increasing total GAG content from elastic cartilage (auricle, epiglottis) to fibrocartilage (meniscus) to hyaline cartilage (nose, knee joint). Analysis of aggregate modulus showed nasal and auricular cartilage to have the greatest stiffness, epiglottal and meniscal tissue the lowest, and articular cartilage intermediate. This study illustrates the differences and identifies unique characteristics of the different cartilage subtypes in rabbits. The results provide a baseline of data for generating and evaluating engineered repair cartilage tissue synthesized in vitro or for post-implantation analysis.

Targeting Improves MSC Treatment of Inflammatory Bowel Disease

Inflammatory bowel disease (IBD), which includes Crohns disease and ulcerative colitis, is an inflammatory autoimmune disease characterized by T-cell infiltration to the colon. Mesenchymal stem cells (MSCs) have the potential to rescue IBD owing to their immunosuppressive capabilities and clinical studies have shown positive influence on intestinal graft versus host disease. We demonstrate here a new method to coat MSCs with antibodies against addressins to enhance their delivery to the colon and thereby increase the therapeutic effectiveness. Bioluminescence imaging (BLI) demonstrated that vascular cell adhesion molecule antibody (Ab)-coated MSCs (Ab(VCAM-1)- MSCs) had the highest delivery efficiency to inflamed mesenteric lymph node (MLN) and colon compared to untreated MSCs, Ab(isotype)-MSCs, and Ab(MAdCAM)-MSCs. Therapeutically, when mice with IBD were injected with addressin Ab-coated MSCs, they showed dramatically improved survival rates, higher IBD therapeutic scores, and significantly improved body weight gain compared to mice injected with MSCs only, isotype Ab, free Ab plus MSCs, or vehicle-only controls. These data demonstrate that anti-addressin Ab coating on MSC increased cell delivery to inflamed colon and increased the efficacy of MSC treatment of IBD. This is the first study showing an increased therapeutic efficacy when stem cells are first coated with antibodies specifically target them to inflamed sites.

Imaging of Mesenchymal Stem Cell Transplant by Bioluminescence and PET

Dynamic measurements of infused stem cells generally require animal euthanasia for single-time-point determinations of engraftment. In this study, we used a triple-fusion reporter system for multimodal imaging to monitor human mesenchymal stem cell (hMSC) transplants. Methods: hMSCs were transduced with a triple-fusion reporter, fluc-mrfp-ttk (encoding firefly luciferase, monomeric red fluorescent protein, and truncated herpes simplex virus type 1 sr39 thymidine kinase) by use of a lentiviral vector. Transduced cells were assayed in vitro for the expression of each functional component of the triple-fusion reporter. Transduced and control hMSCs were compared for their potential to differentiate into bone, cartilage, and fat. hMSCs expressing the reporter were then loaded into porous, fibronectin-coated ceramic cubes and subcutaneously implanted into NOD-SCID mice along with cubes that were loaded with wild-type hMSCs and empty cubes. Mice were imaged repeatedly over 3 mo by bioluminescence imaging (BLI), and selected animals underwent CT and PET imaging. Results: Osteogenic, adipogenic, and chondrogenic potential assays revealed retained differentiation potentials between transduced and wild-type hMSCs. Signals from the cubes loaded with reporter-transduced hMSCs were visible by BLI over 3 mo. There was no signal from the empty or wild-type hMSC–loaded control cubes. PET data provided confirmation of the quantitative estimation of the number of cells at one spot (cube). Cubes were removed from some animals, and histologic evaluations showed bone formation in cubes loaded with either reporter-transduced or wild-type hMSCs, whereas empty controls were negative for bone formation. Conclusion: The triple-fusion reporter approach resulted in a reliable method of labeling stem cells for investigation in small-animal models by use of both BLI and small-animal PET imaging. It has the potential for translation into future human studies with clinical PET.

Clinical-Scale Expansion of a Mixed Population of Bone Marrow-Derived Stem and Progenitor Cells for Potential Use in Bone Tissue Regeneration

Preclinical and clinical studies have demonstrated the ability of bone marrow derived stem and progenitor cells to regenerate many tissues, including bone. Methods to expand or enrich progenitors from bone marrow are common; however, these methods include many steps not amenable to clinical use. A closed automated cell production culture system was developed for clinical‐scale ex vivo production of bone marrow‐derived stem and progenitor cells for hematopoietic reconstitution. The current study tested the ability of this bioreactor system to produce progenitor cells, termed tissue repair cells (TRC), possessing osteogenic potential. Three TRC formulations were evaluated: (a) cells cultured without exogenous cytokines (TRC); (b) cells cultured with exogenous cytokines (TRC‐C); and (c) an adherent subset of TRC‐C (TRC‐CAd). Starting human bone marrow mononuclear cells (BM MNC) and TRC products were characterized for the expression of cell surface markers, in vitro colony forming ability, and in vivo osteogenic potential. Results showed significant expansion of mesenchymal progenitors (CD90+, CD105+, and CD166+) in each TRC formulation. In vivo bone formation, measured by histology, was highest in the TRC group, followed by TRC‐CAd and TRC‐C. The TRC product outperformed starting BM MNC and had equivalent bone forming potential to purified MSCs at the same cell dose. Post hoc analysis revealed that the presence of CD90+, CD105+, and CD166+ correlated strongly with in vivo bone formation scores (r2 > .95). These results demonstrate that this bioreactor system can be used to generate, in a single step, a population of progenitor cells with potent osteogenic potential.

Fabrication of a Neotrachea Using Engineered Cartilage

Objectives: Surgical management of long‐segment tracheal stenosis is an ongoing problem. Many types of tracheal prostheses have been tried but with limited success because of immune rejection, graft ischemia, or restenosis. Tissue engineered cartilage may offer a solution to this problem, although scaffolds, which are currently often used for support, can lead to biocompatibility problems. This study investigated the feasibility of scaffold‐free cartilage to tissue engineer a vascularized neotrachea in rabbits.

Transcriptional profiling of human mesenchymal stem cells transduced with reporter genes for imaging

Mesenchymal stem cells (MSCs) can differentiate into osteogenic, adipogenic, chondrogenic, myocardial, or neural lineages when exposed to specific stimuli, making them attractive for tissue repair and regeneration. We have used reporter gene-based imaging technology to track MSC transplantation or implantation in vivo. However, the effects of lentiviral transduction with the fluc-mrfp-ttk triple-fusion vector on the transcriptional profiles of MSCs remain unknown. In this study, gene expression differences between wild-type and transduced hMSCs were evaluated using an oligonucleotide human microarray. Significance Analysis of Microarray identified differential genes with high accuracy; RT-PCR validated the microarray results. Annotation analysis showed that transduced hMSCs upregulated cell differentiation and antiapoptosis genes while downregulating cell cycle, proliferation genes. Despite transcriptional changes associated with bone and cartilage remodeling, their random pattern indicates no systematic change of crucial genes that are associated with osteogenic, adipogenic, or chondrogenic differentiation. This correlates with the experimental results that lentiviral transduction did not cause the transduced MSCs to lose their basic stem cell identity as demonstrated by osteogenic, chondrogenic, and adipogenic differentiation assays with both transduced and wild-type MSCs, although a certain degree of alterations occurred. Histological analysis demonstrated osteogenic differentiation in MSC-loaded ceramic cubes in vivo. In conclusion, transduction of reporter genes into MSCs preserved the basic properties of stem cells while enabling noninvasive imaging in living animals to study the biodistribution and other biological activities of the cells.

Hyaluronan-based scaffolds to tissue-engineer cartilage implants for laryngotracheal reconstruction

Objectives: Donor site morbidity, including pneumothorax, can be a considerable problem when harvesting cartilage grafts for laryngotracheal reconstruction (LTR). Tissue‐engineered cartilage may offer a solution to this problem. This study investigated the feasibility of using Hyalograft C combined with autologous chondrocytes to tissue engineer cartilage grafts for LTR in rabbits.