(50b) Synthesis and Characterization of Poly(L-Lactide) Networks as in-Situ Crosslinkable Scaffolds for Guided Tissue Regeneration

Poly(lactic acid) (PLA) and its copolymers with
glycolic acid (PLGA) are used in a variety of orthopedic applications as bone fixative, suture reinforcement, and as scaffolds
for cell transplantation and guided regeneration. PLGA copolymers are
semi-crystalline biocompatible copolymers and FDA approved for certain clinical
applications. PLA and PLGA are generally used as preformed scaffolds or foams
because casting from organic solvents or from melts at high temperature are
required for fabrication. We have
developed novel unsaturated PLA, PGA, and PLGA macromers to overcome the use of
solvents or high temperature in fabrication of PLGA constructs. These novel
macromers can be used as injectable in-situ crosslinkable scaffolds in a
variety of applications in guided tissue regeneration.

Poly(L-lactide) unsaturated macromers were synthesized
by condensation polymerization of fumaric acid, a substance that occurs
naturally in the Kreb's cycle, with relatively short lactide or glycolide
chains. Difunctional hydroxyl terminated short lactide or glyoclide chains were
first synthesized by melt ring-opening polymerization of L-lactide (LA) or
glycolide monomer with diethylene glycol (DEG) as the initiator and tin
II-ethyl hexanoate as the catalyst. The molar ratio of LA to DEG was varied
from 10 to 30 to produce low molecular weight PLA (LMWPLA) chains with number
average molecular weights (M_n) in the range of 1000 to 4000 Dalton. The synthesized
LMWPLA was characterized by ¹H-NMR, FTIR, and gel permeation
chromatography (GPC). Next, the LMWPLA was condensed with fumaric acid to form unsaturated
lactide-fumarate (PLAF) copolyesters. The PLAF macromer was characterized by ¹H-NMR,
¹³C-NMR, FTIR, GPC, and differential scanning calorimetry (DSC). Molecular
weight of PLAF depended on that of LMWPLA used in the synthesis. The M_n
of PLAF increased from 4000 to 5200 Dalton as that of LMWPLA increased from 1000 to 2300. The polydispersity
index of PLAF (2.5) was significantly higher than that of LMWPLA (1.6) for all
molecular weights. Copolymerization with Fumaric acid did not significantly
affect the degree of crystallinity of LMWPLA, as measured by WAXD. The melting
point of the semi-crystalline PLAF, measured by DSC, depended on the molecular
weight and fraction of glycolide in the synthesized low molecular weight PLGA
polymer.

Injectable scaffolds were fabricated by free radical
polymerization of PLAF with 1-vinyl-2-pyrrolidinone (VP) to facilitate
crosslinking and sodium chloride (NaCl) crystals as porogen. Benzoyl peroxide
and dimethyl toluidine were used as the free radical initiator and accelerator,
respectively. The polymerizing mixture was injected into a mold, allowed to
crosslink, and the porogen was leached out by soaking the scaffolds in
distilled deionized (DI) water. Pore morphology was investigated with scanning electron
microscopy (SEM). Cell behavior on PLAF surfaces was investigated with rat
neonatal heart fibroblasts. Hearts were dissected, tissue was minced and
digested with collagenase, and fibroblasts were purified by selective
attachment to culture dishes. Fibroblasts showed significant adhesion to PLAF
surfaces and the degree of attachment increased by coating the substrates with
collagen, laminin, or fabronectin. Results demonstrate that the poly(lactide-fumarate)
macromer is potentially useful for fabrication of in-situ crosslinkable
scaffolds for guided tissue regeneration.