A de novo designed beta-hairpin peptide (MAX8), capable of undergoing intramolecular folding and consequent intermolecular self-assembly into a cytocompatible hydrogel, has been studied. A combination of small angle neutron scattering (SANS) and cryogenic-transmission electron microscopy (cryo-TEM) have been used to quantitatively investigate the MAX8 nanofibrillar hydrogel network morphology. A change in the peptide concentration from 0.5 to 2 wt% resulted in a denser fibrillar network as revealed via SANS by a change in the high q (q = (4 pi/lambda) x sin (theta/2), where lambda = wavelength of incident neutrons and theta = scattering angle) mass fractal exponent from 2.5 to 3 and by a decrease in the measured correlation length from 23 to 16 A. A slope of -4 in the USANS regime indicates well-defined gel microporosity, an important characteristic for cellular substrate applications. These changes, both at the network as well as the individual fibril lengthscales, can be directly visualized in situ by cryo-TEM. Fibrillar nanostructures and network properties are directly related to bulk hydrogel stiffness via oscillatory rheology. Preliminary cell viability and anchorage studies at varying hydrogel stiffness confirm cell adhesion at early stages of cell culture within the window of stiffness investigated. Knowledge of the precise structure spanning length scales from the nanoscale up to the microscale can help in the formation of future, specific structure-bioproperty relationships when studying in vitro and in vivo behavior of these new peptide scaffolds.
Nanoparticles of ~10 nm in diameter made with chitosan or lactic acid-grafted chitosan were developed for high drug loading and prolonged drug release. A drug encapsulation efficiency of 92% and a release rate of 28% from chitosan nanoparticles over a 4-week period were demonstrated with bovine serum protein. To further increase drug encapsulation, prolong drug release, and increase chitosan solubility in solution of neutral pH, chitosan was modified with lactic acid by grafting D,L-lactic acid onto amino groups in chitosan without using a catalyst. The lactic acid-grafted chitosan nanoparticles demonstrated a drug encapsulation efficiency of 96% and a protein release rate of 15% over 4 weeks. With increased protein concentration, the drug encapsulation efficiency decreased and drug release rate increased. Unlike chitosan, which is generally soluble only in acid solution, the chitosan modified with lactic acid can be prepared from solutions of neutral pH, offering an additional advantage of allowing proteins or drugs to be uniformly incorporated in the matrix structure with minimal or no denaturization.
Regeneration of mammalian digit tips is well described; however, associated cellular or molecular events have not been studied in humans. We describe an in vitro human fetal model of response to digit tip amputation, and report expression of the transcription repressor Msx1 in the developing and regrowing human digit tip. Human fetal digits from specimens ranging from 53 to 117 days' estimated gestational age (EGA) were cultured in a defined serum-free medium with supplemented oxygen for time periods from 4 days to 4 weeks. Histology and immunohistochemistry were performed on paired control and tip-amputated digits. Regrowing tissue covered the cut end of the distal phalanx in digits up to 80 days' EGA. Msx1 expression was detected beneath the nail field in control digits to at least 70 days' EGA and at the regrowing tip of 57-day digits at 4 and 7 days post-amputation. Our results show that human fetal digits regrow tissue in vitro in response to tip amputation. This process appears spatially associated with Msx1 expression. Msx1 expression appears increased at the regrowing tip of 57-day digits by 4 days after amputation.
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