Sanjay Dhar

Osteoblastic phenotype expression of MC3T3-E1 cells cultured on polymer surfaces.

Background: Current efforts in bone tissue engineering have as one focus the search for a scaffold material that will support osteoblast proliferation, matrix mineralization, and, ultimately, bone formation. The goal is to develop a bone substitute that is functionally equivalent to autograft bone. Previously published reports have shown that osteoblasts exhibit varying rates and degrees of proliferation and mineralization when grown on different surfaces. Methods: This study presents a histologic and biomolecular analysis of MC3T3-E1 murine preosteoblast cells grown on poly(lactide-co-glycolide) (PLGA) versus poly(-caprolactone) (PCL), two commonly studied scaffold polymers. MC3T3-E1 cells were cultured on slides coated with either PLGA or PCL, and on uncoated glass slides as control, with six slides in each group. After 6 weeks in culture, the cells were stained for osteocalcin, alkaline phosphatase activity, and matrix mineralization. In addition, to assess the effects of the surface material on phenotypic expression at the molecular level, MC3T3-E1 cells were cultured on polymer-coated 24-well plates for 4 days, and analyzed by reverse transcription polymerase chain reaction for the expression of osteocalcin and alkaline phosphatase. Results: The results showed that three groups of slides stained positively for osteocalcin at 6 weeks. However, markedly less alkaline phosphatase activity and mineralization were observed on the cells grown on PCL. Real-time polymerase chain reaction assays subsequently revealed decreased expression of both markers by cells cultured on PCL compared with PLGA. Conclusions: These results suggest that PCL does not support the full expression of an osteoblastic phenotype by MC3T3-E1 cells. PCL, therefore, is less desirable as a scaffold polymer in bone tissue engineering in so far as supporting bone formation is concerned. However, because PCL has favorable handling characteristics and strength, modifications of PCL may prompt further investigation.

Release Kinetics of Polymer-bound Bone Morphogenetic Protein-2 and Its Effects on the Osteogenic Expression of Mc3t3-e1 Osteoprecursor Cells

Background: In an effort to augment scaffold performance, additives such as growth factors are under investigation for their ability to optimize the “osteopotential” of synthetic polymer scaffolds. In parallel research, bone morphogenetic protein-2 (BMP-2), a growth factor that initiates bone formation, has been locally delivered to augment fracture healing and spinal fusion. The authors hypothesize that BMP-2 can be covalently bound to a polymer substrate, increasing its concentration and bioavailability over longer periods, thus improving the efficacy of the growth factor and subsequently the bony matrix production. It would remain bound longer when compared with published controls. This prolonged binding would then increase the bioavailability of the growth factor and thus increase bony matrix production over a longer interval. Methods: Mouse preosteoblast MC3T3-E1 cells were cultured on poly(lactic-co-glycolic acid) and polycaprolactone polymer disks covalently bound with BMP-2 to assess the progression and quality of osteogenesis. Covalent binding of BMP-2 to each polymer was visualized by immunohistochemical analysis of polymer-coated microscope slides. The quantity of covalently bound BMP-2 was determined using enzyme-linked immunosorbent assay. Results: Polymerase chain reaction results showed elevated expression levels for alkaline phosphatase and osteocalcin genes. BMP-2 was released from polycaprolactone over 2 weeks, with 86 percent remaining covalently bound, in contrast to 93 percent retained by poly(lactic-co-glycolic acid). Conclusions: BMP-2, proven to alter polymer osteogenicity, remained bound to poly(lactic-co-glycolic acid), which may render poly(lactic-co-glycolic acid) an ideal choice as a polymer for scaffold-based bone tissue engineering using growth factor delivery.

Human embryonic kidney cells (HEK-293 cells): characterization and dose-response relationship for modulated release of nerve growth factor for nerve regeneration.

The development of engineered constructs to bridge nerve gaps may hold the key to improved functional outcomes in the repair of injured peripheral nerves. These constructs must be rendered bioactive by providing the growth factors required for successful peripheral nerve regeneration. Previous studies demonstrated that harvested human and rat dermal fibroblasts could be genetically engineered to release nerve growth factor (NGF) both in vitro and in vivo. The use of fibroblasts, however, has the potential to cause scarring, and the expression of NGF from those cells was transient. To overcome these potential difficulties, human embryonic kidney cells were modified for use with the ecdysone-inducible mammalian expression system. These cells (hNGF-EcR-293) have been engineered and regulated to secrete human NGF in response to the ecdysone analogue ponasterone A. HEK-293 cells were transfected with human NGF cDNA with the ecdysone-inducible mammalian expression system (Invitrogen, Carlsbad, Calif.). Stable clones were then selected. Ponasterone A, an analogue of ecdysone, was used as the inducing agent. The secretion of NGF into the medium was analyzed with two different methods. After 24 hours of exposure to the inducing agent, cell medium was transferred to PC-12 cells seeded in 12-well plates, for determination of whether the secreted NGF was bioactive. Medium from untreated or ponasterone A-treated hNGF-EcR-293 cells was deemed bioactive on the basis of its ability to induce PC-12 cell differentiation. The concentrations of secreted NGF were also quantified with an enzyme-linked immunosorbent assay, in triplicate. NGF production was measured in successive samples of the same medium during a 9-day period, with maximal release of 9.05 ± 2.6 ng/ml at day 9. Maximal NGF production was 8.46 ± 2.1 pg/103 cells at day 9. These levels were statistically significantly different from levels in noninduced samples (p ≤ 0.05). Differences in NGF secretion with the three different concentrations of ponasterone A (1, 2, and 3 &mgr;M) were not statistically significant. PC-12 cells exposed to medium from induced transfected HEK-293 cells demonstrated markedly higher levels of differentiation, compared with control levels, indicating bioactive protein secretion. It was demonstrated that this regulated delivery system could secrete bioactive NGF for up to 9 days and might be useful for in vivo applications. This regulated delivery system should be useful for tissue-engineered nerve constructs.

Pharmacologic enhancement of rat skin flap survival with topical oleic acid.

This study was instituted to investigate in a rat model the effect of topical coadministration of the penetration enhancer oleic acid (10% by volume) and RIMSO-50 (medical grade dimethyl sulfoxide, 50% by volume) on rat skin flap survival. A rectangular abdominal skin flap (2.5 × 3 cm) was surgically elevated over the left abdomen in 40 nude rats. The vein of the flaps neurovascular pedicle was occluded by placement of a microvascular clip, and the flap was resutured with 4-0 Prolene to its adjacent skin. At the end of 8 hours, the distal edge of the flap was reincised to gain access to the clips and the clips were removed. After resuturing of the flaps distal edge to its adjacent skin, the 40 flaps were randomly divided into four groups. Group 1 (control) flaps were treated with 5 g of saline, group 2 (dimethyl sulfoxide) flaps were treated with 2.7 g of dimethyl sulfoxide (50% by volume), group 3 flaps (oleic acid) were topically treated with 0.45 g of oleic acid (10% by volume), and group 4 (dimethyl sulfoxide plus oleic acid) flaps were treated with a mixture of 0.45 g of oleic acid (10% by volume) and 2.7 g of dimethyl sulfoxide (50% by volume) diluted in saline. Each flap was topically treated with 5 ml of drug-soaked gauze for 1 hour immediately after clip removal to attenuate reperfusion injury. Thereafter, drug was applied topically once daily for 4 more days. Digital photographs of each flap were then taken on day 6 and the flaps were then harvested. The percentage of skin survival in each flap was determined by computerized morphometry and planimetry. The mean surviving area of group 3 (oleic acid–treated flaps) was 23.60 ± 4.19 percent and was statistically higher than that in group 1 (control, saline-treated flaps) at 7.20 ± 2.56 percent. The mean surviving area of group 2 (dimethyl sulfoxide–treated flaps) at 18.00 ± 5.23 percent and group 4 (oleic acid–and dimethyl sulfoxide-treated flaps) at 9.90 ± 3.44 percent did not achieve statistically higher mean surviving areas than controls. A topical solution of oleic acid (10% by volume) caused a statistically significant increase in the survival of rat abdominal skin flaps relative to controls. Dimethyl sulfoxide and the two experimental drugs together did not increase the percentage of flap survival when given as a single 5-ml dose released from a surgical sponge at reperfusion for 1 hour and then daily for a total of 5 days. The reasons for the lack of response are unknown but may have included the technical difficulty of delivering an adequate dose of dimethyl sulfoxide topically and immiscibility between dimethyl sulfoxide and oleic acid. Further studies may be warranted.

Inducible nerve growth factor delivery for peripheral nerve regeneration in vivo.

Background: HEK-293 cells can be genetically modified to release and regulate nerve growth factor (NGF) in vitro. The aim of this study was to evaluate the impact of this NGF delivery system on peripheral nerve regeneration in vivo. Methods: HEK-293 cells were transfected with an ecdysone receptor, NGF cDNA, and herpes simplex virus–thymidine kinase suicide vector. NGF production is induced by ponasterone A and stopped by ganciclovir. A 13-mm sciatic nerve gap was bridged with Silastic conduits in 120 nude rats, and transfected HEK-293 cells were added, induced, and boostered to secrete bioactive NGF. Results: The induction of the cell line and additional booster with ponasterone A demonstrated significantly higher levels of bioactive NGF, enhanced macroscopic nerve growth, improved functional recovery, and histologic regeneration when compared with control groups after 7, 14, and 21 days, and 2 and 4 months. The treatment with ganciclovir resulted in suppression of the NGF production and decreased functional and histologic outcomes. Conclusions: Transfected HEK-293 cells can be regulated to inducibly produce bioactive NGF in vivo over prolonged periods. This tissue-engineered nerve construct including the NGF delivery system is able to improve peripheral nerve regeneration and functional recovery and appears to be superior to nerve isografts.

Correlation of rapid phenotypic changes and insulin production of differentiated human adipose tissue-derived stem cells.

The study investigates adipose tissue-derived stem cells (ADSCs) to differentiate into insulin-secreting cells in vitro. ADSCs were exposed to differentiation medium or control medium. Culture medium was harvested for quantification of insulin and cells were classified into stages of differentiation ranging from normal appearance to islet-like clusters. Morphologic analysis demonstrated marked phenotypic changes towards the islet cell lineage. Thirty-six percent of cells exposed to differentiation medium had progressed to morphologic end-stage after 15 days. Chemiluminescence of the culture medium determined that insulin secretion by differentiated cells progressively increased, reaching a maximum at day 7. Insulin secretion by control cells was significantly less at all time points. A high correlation of secreted insulin and the presence of stage 3 cells were observed throughout the entire experiment. ADSCs can be differentiated into insulin-secreting cells in response to defined culture conditions. The secreted insulin significantly correlates with phenotypic changes throughout the differentiation process.