Medium chain length polyhydroxyalkanoates as potential matrix materials for peripheral nerve regeneration.

Polyhydroxyalkanoates are natural, biodegradable, thermoplastic and sustainable polymers with a huge potential in fabrication of bioresorbable implantable devices for tissue engineering. We describe a comparative evaluation of three medium chain length polyhydroxyalkanoates (mcl-PHAs), namely poly(3-hydroxyoctanoate), poly(3-hydroxyoctanoate-co-3-hydoxydecanoate) and poly(3-hydroxyoctanoate-co-3-hydroxydecanoate-co-3-hydroxydodecanoate), one short chain length polyhydroxyalkanoate, poly(3-hydroxybutyrate), P(3HB) and synthetic aliphatic polyesters (polycaprolactone and polylactide) with a specific focus on nerve regeneration, due to mechanical properties of mcl-PHAs closely matching nerve tissues. In vitro biological studies with NG108-15 neuronal cell and primary Schwann cells did not show a cytotoxic effect of the materials on both cell types. All mcl-PHAs supported cell adhesion and viability. Among the three mcl-PHAs, P(3HO-co-3HD) exhibited superior properties with regards to numbers of cells adhered and viable cells for both cell types, number of neurite extensions from NG108-15 cells, average length of neurite extensions and Schwann cells. Although, similar characteristics were observed for flat P(3HB) surfaces, high rigidity of this biomaterial, and FDA-approved polymers such as PLLA, limits their applications in peripheral nerve regeneration. Therefore, we have designed, synthesized and evaluated these materials for nerve tissue engineering and regenerative medicine, the interaction of mcl-PHAs with neuronal and Schwann cells, identifying mcl-PHAs as excellent materials to enhance nerve regeneration and potentially their clinical application in peripheral nerve repair.

Dually crosslinked injectable alginate-based graft copolymer thermoresponsive hydrogels as 3D printing bioinks for cell spheroid growth and release.

In this work a dual crosslinked network based on sodium alginate graft copolymer, bearing poly(N-isopropylacrylamide-co-N-tert-butylacrylamide) P(NIPAM-co-NtBAM) side chains was developed and examined as a shear thinning soft gelating bioink. The copolymer was found to undergo a two-step gelation mechanism; in the first step a three-dimensional (3D) network is formed through ionic interactions between the negatively ionized carboxylic groups of the alginate backbone and the positive charges of Ca2+ divalent cations, according to the "egg-box" mechanism. The second gelation step occurs upon heating which triggers the hydrophobic association of the thermoresponsive P(NIPAM-co-NtBAM) side chains, increasing the network crosslinking density in a highly cooperative manner. Interestingly, the dual crosslinking mechanism resulted in a five-to-eight-fold improvement of the storage modulus implying reinforced hydrophobic crosslinking above the critical thermo-gelation temperature which is further boosted by the ionic crosslinking of the alginate backbone. The proposed bioink could form arbitrary geometries under mild 3D printing conditions. Last, it is demonstrated that the proposed developed bioink can be further utilized as bioprinting ink and showcased its ability to promote human periosteum derived cells (hPDCs) growth in 3D and their capacity to form 3D spheroids. In conclusion, the bioink, owing its ability to reverse thermally the crosslinking of its polymer network, can be further utilized for the facile recovery of the cell spheroids, implying its promising potential use as cell spheroid-forming template bionk for applications in 3D biofabrication.

Preclinical study of peripheral nerve regeneration using nerve guidance conduits based on polyhydroxyalkanaotes.

Nerve guidance conduits (NGCs) are used as an alternative to the "gold standard" nerve autografting, preventing the need for surgical intervention required to harvest autologous nerves. However, the regeneration outcomes achieved with the current NGCs are only comparable with autografting when the gap is short (less than 10 mm). In the present study, we have developed NGCs made from a blend of polyhydroxyalkanoates, a family of natural resorbable polymers. Hollow NGCs made from a 75:25 poly(3-hydroxyoctanoate)/poly(3-hydroxybutyrate) blend (PHA-NGCs) were manufactured using dip-molding. These PHA-NGCs showed appropriate flexibility for peripheral nerve regeneration. In vitro cell studies performed using RT4-D6P2T rat Schwann cell line confirmed that the material is capable of sustaining cell proliferation and adhesion. PHA-NGCs were then implanted in vivo to repair 10 mm gaps of the median nerve of female Wistar rats for 12 weeks. Functional evaluation of the regenerated nerve using the grasping test showed that PHA-NGCs displayed similar motor recovery as the autograft, starting from week 7. Additionally, nerve cross-sectional area, density and number of myelinated cells, as well as axon diameter, fiber diameter, myelin thickness and g-ratio obtained using the PHA-NGCs were found comparable to an autograft. This preclinical data confirmed that the PHA-NGCs are indeed highly promising candidates for peripheral nerve regeneration.

Antimicrobial Materials with Lime Oil and a Poly(3-hydroxyalkanoate) Produced via Valorisation of Sugar Cane Molasses.

A medium chain-length polyhydroxyalkanoate (PHA) was produced by Pseudomonas mendocina CH50 using a cheap carbon substrate, sugarcane molasses. A PHA yield of 14.2% dry cell weight was achieved. Chemical analysis confirmed that the polymer produced was a medium chain-length PHA, a copolymer of 3-hydroxyoctanoate and 3-hydroxydecanoate, P(3HO-co-3HD). Lime oil, an essential oil with known antimicrobial activity, was used as an additive to P(3HO-co-3HD) to confer antibacterial properties to this biodegradable polymer. The incorporation of lime oil induced a slight decrease in crystallinity of P(3HO-co-3HD) films. The antibacterial properties of lime oil were investigated using ISO 20776 against Staphylococcus aureus 6538P and Escherichia coli 8739, showing a higher activity against the Gram-positive bacteria. The higher activity of the oil against S. aureus 6538P defined the higher efficiency of loaded polymer films against this strain. The effect of storage on the antimicrobial properties of the loaded films was investigated. After one-year storage, the content of lime oil in the films decreased, causing a reduction of the antimicrobial activity of the materials produced. However, the films still possessed antibacterial activity against S. aureus 6538P.