Synthesis and in vitro biocompatibility of injectable polyurethane foam scaffolds.

The development of therapeutics for orthopedic clinical indications exploiting minimally invasive surgical techniques has substantial benefits, especially for treatment of fragility fractures in the distal radius of osteoporotics and vertebral compression fractures. We have designed six formulations of injectable polyurethane foams to address these clinical indications. The polyurethanes were prepared by mixing two liquid components and injecting the reactive liquid mixture into a mold where it hardens in situ. Porous polyurethane foams were synthesized from lysine methyl ester diisocyanate, a poly(epsilon-caprolactone-co-glycolide) triol, a tertiary amine catalyst, anionic and non-ionic stabilizers, and a fatty acid pore opener. The rise time of the foams varied from 8-20 min. The porosity was approximately 95% and the pores varied in size from 100-1000 microm. The polyurethane foams supported attachment of viable (>95%) MG-63 cells under dynamic seeding conditions. We anticipate compelling opportunities will be available as a consequence of the favorable biological and physical properties of the injectable polyurethane foams.

Synthesis of biocompatible segmented polyurethanes from aliphatic diisocyanates and diurea diol chain extenders.

Many polyurethane elastomers display excellent mechanical properties and adequate biocompatibility. However, many medical-grade polyurethanes are prepared from aromatic diisocyanates and can degrade in vivo to carcinogenic aromatic diamines, although the question of whether the concentrations of these harmful degradation products attain physiologically relevant levels is currently unresolved and strongly debated. It is therefore desirable to synthesize new medical-grade polyurethanes from less toxic aliphatic diisocyanates. In this paper, biocompatible segmented polyurethane elastomers were synthesized from aliphatic diisocyanates (1,4-diisocyanatobutane (BDI) and lysine methyl ester diisocyanate (LDI)), novel diurea diol chain extenders based on tyrosine and tyramine, and a model poly(ethylene glycol) (PEG) diol soft segment. The objectives were to design a hard segment similar in structure to that of MDI-based polyurethanes and also investigate the effects of systematic changes in structure on mechanical and biological properties. The non-branched, symmetric polyurethane prepared from BDI and a tyramine-based chain extender had the highest modulus at 37 degrees C. Introduction of symmetric short-chain branches (SCBs) incorporated in the tyrosine-based chain extender lowered the modulus by an order of magnitude. Polyurethanes prepared from LDI were soft polymers that had a still lower modulus due to the asymmetric SCBs that hindered hard segment packing. Polyurethanes prepared from tyramine and tyrosine chain extenders thermally degraded at temperatures ranging from 110 to 150 degrees C, which are lower than that reported previously for phenyl urethanes. All four polyurethanes supported the attachment, proliferation, and high viability of MG-63 human osteoblast-like cells in vitro. Therefore, the non-cytotoxic chemistry of these polyurethanes make them good candidates for further development as biomedical implants.

Multi-channeled biodegradable polymer/CultiSpher composite nerve guides.

Innovative methods to fabricate porous, biodegradable conduits were developed to produce nerve guides with multiple longitudinally aligned channels. The geometry of the nerve guide's channels was designed to be appropriate for harboring neurite extension. Both the coated mandrel and mandrel adhesion techniques permit flexibility in the number of channels, channel organization, and channel diameters. In this study, the composite nerve guides were comprised of poly(caprolactone) (PCL) and porous collagen-based beads (CultiSphers). The incorporation of the collagenous beads results in enhanced cortical neuron adhesion, viability, and neurite extension as compared to PCL alone. Additionally, Schwann cell studies indicated that the PCL/CultiSpher composite is a suitable substrate for cell adhesion. Mechanical properties of the PCL/CultiSpher material and in vitro degradation rates indicate the potential usefulness of this novel composite for use in the fabrication of nerve guides.

Using PC12 cells to evaluate poly(caprolactone) and collagenous microcarriers for applications in nerve guide fabrication.

Tissue-engineered nerve guides can provide mechanical support as well as chemical stimulation for nerve regeneration. PC12 cells were used to test the novel combination of poly(caprolactone) (PCL) and macroporous collagen-based microcarriers (CultiSphers) as an initial phase in the fabrication of multichanneled nerve guides. Laminin-coated PCL was an effective matrix for the attachment, proliferation, and viability of PC12 cells, relative to uncoated PCL. PC12 cells attached to laminin-coated PCL and extended neurites when cultured in the presence of nerve growth factor (NGF). PC12 cells attached and proliferated on CultiSphers and extended neurites in response to NGF. A novel PCL/CultiSpher composite material also supported PC12 attachment and proliferation and provides a potentially useful material for the fabrication of synthetic nerve guides.

Collagenous microbeads as a scaffold for tissue engineering with adipose-derived stem cells.

BACKGROUND: Standard approaches to soft-tissue reconstruction include autologous tissue flaps and alloplastic implants. Both of these approaches have disadvantages, including donor-site morbidity, implant migration, and foreign body reaction. Autologous fat transplantation, with a minimally invasive cannula harvest, has lower donor-site morbidity than tissue flaps do, but there is an unpredictable degree of resorption of the transplanted fat over time. Adipose-derived stem cells isolated from harvested fat are better able to withstand the mechanical trauma from the suction cannula and may allow for improved cell survival and generation of new fat tissue after transfer to another anatomic site. The authors hypothesized that porous collagenous microbeads (CultiSphers; Sigma, St. Louis, Mo.) could be useful as injectable cell delivery vehicles for adipose-derived stem cells. This strategy would allow induction of differentiation ex vivo and precise placement of cells and scaffold in a tissue bed. The objective of this study was to assess the ability of the stem cells to proliferate and differentiate on these microbeads. METHODS: Adipose-derived stem cells were isolated from discarded human adipose tissue and cultured on porous collagenous microbeads in a stirred bioreactor (spinner flask). The cells attached and proliferated on the microbeads and maintained high viability over several weeks of culture. RESULTS: When exposed to adipogenic or osteogenic medium, the cells differentiated into adipocytes and osteoblasts, respectively, while attached to the microbeads. CONCLUSION: Collagenous microbeads are a favorable scaffold for adipose-derived stem cells, allowing ex vivo proliferation and differentiation on particles that are small enough to be injected.

Comparison of biodegradable conduits within aged rat sciatic nerve defects.

BACKGROUND: Considering that little is known about the peripheral nerve regenerative capacity of elderly patients, the authors studied nerve regenerative capacity in aged rats and compared the effect of three synthetic nerve guides with different material characteristics and porosity. The authors hypothesized that the use of a biodegradable composite nerve guide (CultiGuides) would promote nerve regeneration and functional recovery in a manner similar to treatment with autografts or U.S. Food and Drug Administration-approved polyglycolic acid Neurotubes in an aged rat sciatic nerve defect model. METHODS: Aged Sprague-Dawley rats (11 months old) underwent a 1-cm sciatic nerve resection in the right leg [group 1, control (contralateral leg samples), n = 10; group 2, negative (nerve gap defect), n = 6; group 3, autograft, n = 10; group 4, polycaprolactone, n = 10; group 5, CultiGuides, n = 10; and group 6, Neurotube, n = 10]. RESULTS: After 12 weeks, the negative group did not demonstrate any nerve regeneration. In the regenerated and distal nerve, all treated groups had increased myelinated areas compared with the negative control. In the regenerated nerve, there was a significant increase in myelination in the Neurotube group compared with the polycaprolactone group (p < 0.001). However, in the distal nerve, there were no differences among the treatment groups. Walking track analyses and gastrocnemius muscle weight ratios were not different among treatment groups 3 through 6. CONCLUSIONS: The results showed differences in myelination; Neurotubes promoted the highest degree of myelination (p < 0.001) as compared with all groups. The authors found no improvement in function of the repaired nerve as measured by percentage of autotomy, the sciatic function index, and gastrocnemius muscle weight. No group was able to recover function in this aged model.

Melatonin enhances alkaline phosphatase activity in differentiating human adult mesenchymal stem cells grown in osteogenic medium via MT2 melatonin receptors and the MEK/ERK (1/2) signaling cascade.

The goals of this study were to determine (a) if melatonin enhances human adult mesenchymal stem cell (hAMSC) differentiation into osteoblasts as assessed by measuring alkaline phosphatase (ALP) enzyme activity, and (b) identify potential signal transduction pathways that mediate this process. ALP activity significantly increased in hAMSCs following a 10-day incubation in osteogenic medium, relative to hAMSCs incubated in basal growth medium alone. Melatonin (50 nm), added in combination with the osteogenic medium, significantly increased ALP activity relative to osteogenic medium alone. Co-exposure of hAMSCs to osteogenic medium supplemented with melatonin and either pertussis toxin or the melatonin receptor antagonists, luzindole or 4P-PDOT (MT2 receptor selective), inhibited the melatonin-induced increase in ALP activity, indicating the involvement of melatonin receptors, in particular, MT2 receptors. Assessment of melatonin receptor function following exposure to osteogenic medium containing either vehicle or melatonin produced dichotomous results. That is, if the differentiation of hAMSCs into an osteoblast was induced by osteogenic medium alone, then 2-[125I]-iodomelatonin binding and melatonin receptor function increased. However, examination of melatonin receptor function following chronic melatonin exposure, an exposure that resulted in a 50% enhancement in ALP activity, revealed that these receptors were desensitized. This was reflected by a complete loss in specific 2-[125I]-iodomelatonin binding as well as melatonin efficacy to inhibit forskolin-induced cAMP accumulation. Further characterization of the mechanisms underlying melatonin's effects on these differentiation processes revealed that MEK (1/2) and ERK (1/2), epidermal growth factor receptors, metalloproteinase and clathrin-mediated endocytosis were essential while PKA was not. Our results are consistent with a role for melatonin in osteoblast differentiation. If so, then, the decrease in plasma melatonin levels observed in humans during late adulthood may further enhance susceptibility to osteoporosis.

Therapeutic treatments potentially mediated by melatonin receptors: potential clinical uses in the prevention of osteoporosis, cancer and as an adjuvant therapy.

Melatonin's therapeutic potential is grossly underestimated because its functional roles are diverse and its mechanism(s) of action are complex and varied. Melatonin produces cellular effects via a variety of mechanisms in a receptor independent and dependent manner. In addition, melatonin is a chronobiotic agent secreted from the pineal gland during the hours of darkness. This diurnal release of melatonin impacts the sensitivity of melatonin receptors throughout a 24-hr period. This changing sensitivity probably contributes to the narrow therapeutic window for use of melatonin in treating sleep disorders, that is, at the light-to-dark (dusk) or dark-to-light (dawn) transition states. In addition to the cyclic changes in melatonin receptors, many genes cycle over the 24-hr period, independent or dependent upon the light/dark cycle. Interestingly, many of these genes support a role for melatonin in modulating metabolic and cardiovascular physiology as well as bone metabolism and immune function and detoxification of chemical agents and cancer reduction. Melatonin also enhances the actions of a variety of drugs or hormones; however, the role of melatonin receptors in modulating these processes is not known. The goal of this review is to summarize the evidence related to the utility of melatonin as a therapeutic agent by focusing on its other potential uses besides sleep disorders. In particular, its use in cancer prevention, osteoporosis and, as an adjuvant to other therapies are discussed. Also, the role that melatonin and, particularly, its receptors play in these processes are highlighted.