Bone Grafts Engineered from Human Adipose-Derived Stem Cells in Perfusion Bioreactor Culture

We report engineering of half-centimeter–sized bone constructs created in vitro using human adipose-derived stem cells (hASCs), decellularized bone scaffolds, and perfusion bioreactors. The hASCs are easily accessible, can be used in an autologous fashion, are rapidly expanded in culture, and are capable of osteogenic differentiation. hASCs from four donors were characterized for their osteogenic capacity, and one representative cell population was used for tissue engineering experiments. Culture-expanded hASCs were seeded on fully decellularized native bone scaffolds (4 mm diameter × 4 mm thick), providing the necessary structural and mechanical environment for osteogenic differentiation, and cultured in bioreactors with medium perfusion. The interstitial flow velocity was set to a level necessary to maintain cell viability and function throughout the construct volume (400 μm/s), via enhanced mass transport. After 5 weeks of cultivation, the addition of osteogenic supplements (dexamethasone, sodium-β-glycerophosphate, and ascorbic acid-2-phosphate) to culture medium significantly increased the construct cellularity and the amounts of bone matrix components (collagen, bone sialoprotein, and bone osteopontin). Medium perfusion markedly improved the distribution of cells and bone matrix in engineered constructs. In summary, a combination of hASCs, decellularized bone scaffold, perfusion culture, and osteogenic supplements resulted in the formation of compact and viable bone tissue constructs

Platelets and Plasma Proteins Are Both Required to Stimulate Collagen Gene Expression by Anterior Cruciate Ligament Cells in Three-Dimensional Culture

Collagen–platelet (PL)-rich plasma composites have shown in vivo potential to stimulate anterior cruciate ligament (ACL) healing at early time points in large animal models. However, little is known about the cellular mechanisms by which the plasma component of these composites may stimulate healing. We hypothesized that the components of PL-rich plasma (PRP), namely the PLs and PL-poor plasma (PPP), would independently significantly influence ACL cell viability and metabolic activity, including collagen gene expression. To test this hypothesis, ACL cells were cultured in a collagen type I hydrogel with PLs, PPP, or the combination of the two (PRP) for 14 days. The inclusion of PLs, PPP, and PRP all significantly reduced the rate of cell apoptosis and enhanced the metabolic activity of fibroblasts in the collagen hydrogel. PLs promoted fibroblast-mediated collagen scaffold contraction, whereas PPP inhibited this contraction. PPP and PRP both promoted cell elongation and the formation of wavy fibrous structure in the scaffolds. The addition of only PLs or only plasma proteins did not significantly enhance gene expression of collagen types I and III but the combination, as PRP, did. Our findings suggest that the addition of both PLs and plasma proteins to collagen hydrogel may be useful in stimulating ACL healing by enhancing ACL cell viability, metabolic activity, and collagen synthesis

The evaluation of a biphasic osteochondral implant coupled with an electrospun membrane in a large animal model

Conventional clinical therapies are unable to resolve osteochondral defects adequately, hence tissue engineering solutions are sought to address the challenge. A biphasic implant which was seeded with Mesenchymal Stem Cells (MSC) and coupled with an electrospun membrane was evaluated as an alternative. This dual phase construct comprised of a Polycaprolactone (PCL) cartilage scaffold and a Polycaprolactone - Tri Calcium Phosphate (PCL - TCP) osseous matrix. Autologous MSC was seeded into the entire implant via fibrin and the construct was inserted into critically sized osteochondral defects located at the medial condyle and patellar groove of pigs. The defect was resurfaced with a PCL - collagen electrospun mesh that served as a substitute for periosteal flap in preventing cell leakage. Controls either without implanted MSC or resurfacing membrane were included. After 6 months, cartilaginous repair was observed with a low occurrence of fibrocartilage at the medial condyle. Osteochondral repair was promoted and host cartilage degeneration was arrested as shown by the superior Glycosaminoglycan (GAG) maintenance. This positive morphological outcome was supported by a higher relative Young's modulus which indicated functional cartilage restoration. Bone in growth and remodeling occurred in all groups with a higher degree of mineralization in the experimental group. Tissue repair was compromised in the absence of the implanted cells or the resurfacing membrane. Moreover healing was inferior at the patellar groove as compared to the medial condyle and this was attributed to the native biomechanical features