Hani A. Awad

Clonal analysis of the differentiation potential of human adipose-derived adult stem cells

Pools of human adipose‐derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell‐based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty‐five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage‐specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron‐like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

Repair of patellar tendon injuries using a cell–collagen composite

Collagen gels were seeded with rabbit bone marrow‐derived mesenchymal stem cells (MSCs) and contracted onto sutures at initial cell densities of 1, 4, and 8 million cells/ml. These MSC–collagen composites were then implanted into full thickness, full length, central defects created in the patellar tendons of the animals providing the cells. These autologous repairs were compared to natural repair of identical defects on the contralateral side. Biomechanical, histological, and morphometric analyses were performed on both repair tissue types at 6, 12, and 26 weeks after surgery. Repair tissues containing the MSC–collagen composites showed significantly higher maximum stresses and moduli than natural repair tissues at 12 and 26 weeks postsurgery. However, no significant differences were observed in any dimensional or mechanical properties of the repair tissues across seeding densities at each evaluation time. By 26 weeks, the repairs grafted with MSC–collagen composites were one‐fourth of the maximum stress of the normal central portion of the patellar tendon with bone ends. The modulus and maximum stress of the repair tissues grafted with MSC–collagen composites increased at significantly faster rates than did natural repairs over time. Unexpectedly, 28% of the MSC–collagen grafted tendons formed bone in the regenerating repair site. Except for increased repair tissue volume, no significant differences in cellular organization or histological appearance were observed between the natural repairs and MSC–collagen grafted repairs. Overall, these results show that surgically implanting tissue engineered MSC–collagen composites significantly improves the biomechanical properties of tendon repair tissues, although greater MSC concentrations produced no additional significant histological or biomechanical improvement.

3D printing of composite calcium phosphate and collagen scaffolds for bone regeneration.

Low temperature 3D printing of calcium phosphate scaffolds holds great promise for fabricating synthetic bone graft substitutes with enhanced performance over traditional techniques. Many design parameters, such as the binder solution properties, have yet to be optimized to ensure maximal biocompatibility and osteoconductivity with sufficient mechanical properties. This study tailored the phosphoric acid-based binder solution concentration to 8.75 wt% to maximize cytocompatibility and mechanical strength, with a supplementation of Tween 80 to improve printing. To further enhance the formulation, collagen was dissolved into the binder solution to fabricate collagen-calcium phosphate composites. Reducing the viscosity and surface tension through a physiologic heat treatment and Tween 80, respectively, enabled reliable thermal inkjet printing of the collagen solutions. Supplementing the binder solution with 1-2 wt% collagen significantly improved maximum flexural strength and cell viability. To assess the bone healing performance, we implanted 3D printed scaffolds into a critically sized murine femoral defect for 9 weeks. The implants were confirmed to be osteoconductive, with new bone growth incorporating the degrading scaffold materials. In conclusion, this study demonstrates optimization of material parameters for 3D printed calcium phosphate scaffolds and enhancement of material properties by volumetric collagen incorporation via inkjet printing.

Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.

A murine segmental femoral bone graft model was used to show the essential role of donor periosteal progenitor cells in bone graft healing. Transplantation of live bone graft harvested from Rosa 26A mice showed that ∼70% of osteogenesis on the graft was attributed to the expansion and differentiation of donor periosteal progenitor cells. Furthermore, engraftment of BMP‐2‐producing bone marrow stromal cells on nonvital allografts showed marked increases in cortical graft incorporation and neovascularization, suggesting that gene‐enhanced, tissue engineered functional periosteum may improve allograft incorporation and repair.

In vitro characterization of mesenchymal stem cell-seeded collagen scaffolds for tendon repair: effects of initial seeding density on contraction kinetics.

Mesenchymal stem cells (MSCs) were isolated from bone marrow, culture-expanded, and then seeded at 1, 4, and 8 million cells/mL onto collagen gel constructs designed to augment tendon repair in vivo. To investigate the effects of seeding density on the contraction kinetics and cellular morphology, the contraction of the cell/collagen constructs was monitored over time up to 72 h in culture conditions. Constructs seeded at 4 and 8 million cells/mL showed no significant differences in their gross appearance and dimensions throughout the contraction process. By contrast, constructs seeded at 1 million cells/mL initially contracted more slowly and their diameters at 72 h were 62 to 73% larger than those seeded at higher densities. During contraction, MSCs reoriented and elongated significantly with time. Implants prepared at higher seeding densities showed more well aligned and elongated cell nuclei after 72 h of contraction. Changes in nuclear morphology of the MSCs in response to physical constraints provided by the contracted collagen fibrils may trigger differentiation pathways toward the fibroblastic lineage and influence the cell synthetic activity. Controlling the contraction and organization of the cells and matrix will be critical for successfully creating tissue engineered grafts.

Chondrogenic differentiation of adipose-derived adult stem cells by a porous scaffold derived from native articular cartilage extracellular matrix.

Adipose-derived adult stem cells (ASCs) have the ability to differentiate into a chondrogenic phenotype in response to specific environmental signals such as growth factors or artificial biomaterial scaffolds. In this study, we examined the hypothesis that a porous scaffold derived exclusively from articular cartilage can induce chondrogenesis of ASCs. Human ASCs were seeded on porous scaffolds derived from adult porcine articular cartilage and cultured in standard medium without exogenous growth factors. Chondrogenesis of ASCs seeded within the scaffold was evident by quantitative RT-PCR analysis for cartilage-specific extracellular matrix (ECM) genes. Histological and immunohistochemical examination showed abundant production of cartilage-specific ECM components-particularly, type II collagen-after 4 or 6 weeks of culture. After 6 weeks of culture, the cellular morphology in the ASC-seeded constructs resembled those in native articular cartilage tissue, with rounded cells residing in the glycosaminoglycan-rich regions of the scaffolds. Biphasic mechanical testing showed that the aggregate modulus of the ASC-seeded constructs increased over time, reaching 150 kPa by day 42, more than threefold higher than that of the unseeded controls. These results suggest that a porous scaffold derived from articular cartilage has the ability to induce chondrogenic differentiation of ASCs without exogenous growth factors, with significant synthesis and accumulation of ECM macromolecules, and the development of mechanical properties approaching those of native cartilage. These findings support the potential for a processed cartilage ECM as a biomaterial scaffold for cartilage tissue engineering. Additional in vivo evaluation is necessary to fully recognize the clinical implication of these observations.

Effects of Transforming Growth Factor β1 and Dexamethasone on the Growth and Chondrogenic Differentiation of Adipose-Derived Stromal Cells

The effects of soluble mediators and medium supplements commonly used to induce chondrogenic differentiation in different cell culture systems were investigated to define their dose-response profiles and potentially synergistic effects on the chondrogenic differentiation of adipose-derived adult stromal (ADAS) cells. Human ADAS cells were suspended within alginate beads and cultured in basal medium with insulin, transferrin, and selenious acid (ITS+) or fetal bovine serum (FBS) and treated with different doses and combinations of TGF-beta1 (0, 1, and 10 ng/mL) and dexamethasone (0, 10, and 100 nM). Cell growth and chondrogenic differentiation were assessed by measuring DNA content, protein and proteoglycan synthesis rates, and proteoglycan accumulation. The combination of ITS+ and TGF-beta1 significantly increased cell proliferation. Protein synthesis rates were increased by TGF-beta1 and dexamethasone in the presence of ITS+ or FBS. While TGF-beta1 significantly increased proteoglycan synthesis and accumulation by 1.5- to 2-fold in the presence of FBS, such effects were suppressed by dexamethasone. In summary, the combination of TGF-beta1 and ITS+ stimulated cell growth and synthesis of proteins and proteoglycans by human ADAS cells. The addition of dexamethasone appeared to amplify protein synthesis but had suppressive effects on proteoglycan synthesis and accumulation.

Influence of oxygen on the proliferation and metabolism of adipose derived adult stem cells

Articular cartilage is an avascular connective tissue that exhibits little intrinsic capacity for repair. Articular cartilage exists in a reduced oxygen (∼5%) environment in vivo; therefore, oxygen tension may be an important factor that regulates the metabolism of chondrocyte progenitors. A number of recent studies have developed tissue engineering approaches for promoting cartilage repair using undifferentiated progenitor cells seeded on biomaterial scaffolds, but little is known about how oxygen might influence these engineered tissues. Human adipose‐derived adult stem (hADAS) cells isolated from the stroma of subcutaneous fat were suspended in alginate beads and cultured in control or chondrogenic media in either low oxygen (5%) or atmospheric oxygen tension (20%) for up to 14 days. Under chondrogenic conditions, low oxygen tension significantly inhibited the proliferation of hADAS cells, but induced a two‐fold increase in the rate of protein synthesis and a three‐fold increase in total collagen synthesis. Low oxygen tension also increased glycosaminoglycan synthesis at certain timepoints. Immunohistochemical analysis showed significant production of cartilage‐associated matrix molecules, including collagen type II and chondroitin‐4‐sulfate. These findings suggest oxygen tension may play an important role in regulating the proliferation and metabolism of hADAS cells as they undergo chondrogenesis, and the exogenous control of oxygen tension may provide a means of increasing the overall accumulation of matrix macromolecules in tissue‐engineered cartilage.

A Perspective: Engineering Periosteum for Structural Bone Graft Healing

Autograft is superior to both allograft and synthetic bone graft in repair of large structural bone defect largely due to the presence of multipotent mesenchymal stem cells in periosteum. Recent studies have provided further evidence that activation, expansion and differentiation of the donor periosteal progenitor cells are essential for the initiation of osteogenesis and angiogenesis of donor bone graft healing. The formation of donor cell-derived periosteal callus enables efficient host-dependent graft repair and remodeling at the later stage of healing. Removal of periosteum from bone autograft markedly impairs healing whereas engraftment of multipotent mesenchymal stem cells on bone allograft improves healing and graft incorporation. These studies provide rationale for fabrication of a biomimetic periosteum substitute that could fit bone of any size and shape for enhanced allograft healing and repair. The success of such an approach will depend on further understanding of the molecular signals that control inflammation, cellular recruitment as well as mesenchymal stem cell differentiation and expansion during the early phase of the repair process. It will also depend on multidisciplinary collaborations between biologists, material scientists and bioengineers to address issues of material selection and modification, biological and biomechanical parameters for functional evaluation of bone allograft healing.

Reduced COX-2 Expression in Aged Mice Is Associated With Impaired Fracture Healing

The cellular and molecular events responsible for reduced fracture healing with aging are unknown. Cyclooxygenase 2 (COX‐2), the inducible regulator of prostaglandin E2 (PGE2) synthesis, is critical for normal bone repair. A femoral fracture repair model was used in mice at either 7–9 or 52–56 wk of age, and healing was evaluated by imaging, histology, and gene expression studies. Aging was associated with a decreased rate of chondrogenesis, decreased bone formation, reduced callus vascularization, delayed remodeling, and altered expression of genes involved in repair and remodeling. COX‐2 expression in young mice peaked at 5 days, coinciding with the transition of mesenchymal progenitors to cartilage and the onset of expression of early cartilage markers. In situ hybridization and immunohistochemistry showed that COX‐2 is expressed primarily in early cartilage precursors that co‐express col‐2. COX‐2 expression was reduced by 75% and 65% in fractures from aged mice compared with young mice on days 5 and 7, respectively. Local administration of an EP4 agonist to the fracture repair site in aged mice enhanced the rate of chondrogenesis and bone formation to levels observed in young mice, suggesting that the expression of COX‐2 during the early inflammatory phase of repair regulates critical subsequent events including chondrogenesis, bone formation, and remodeling. The findings suggest that COX‐2/EP4 agonists may compensate for deficient molecular signals that result in the reduced fracture healing associated with aging.