Effects of annulus defects and implantation of poly(lactic-co-glycolic acid) (PLGA)/fibrin gel scaffolds on nerves ingrowth in a rabbit model of annular injury disc degeneration

Abstract

BackgroundGrowth of nerve fibers has been shown to occur in a rabbit model of intravertebral disc degeneration (IVD) induced by needle puncture. As nerve growth may underlie the process of chronic pain in humans affected by disc degeneration, we sought to investigate the factors underlying nerve ingrowth in a minimally invasive annulotomy rabbit model of IVD by comparing the effects of empty disc defects with those of defects filled with poly(lactic-co-glycolic acid)/fibrin gel (PLGA) plugs.MethodsNew Zealand white rabbits (n = 24) received annular injuries at three lumbar levels (L3/4, L4/5, and L5/6). The discs were randomly assigned to four groups: (a) annular defect (1.8-mm diameter; 4-mm depth) by mini-trephine, (b) annular defect implanted with a PLGA scaffold containing a fibrin gel, (c) annular puncture by a 16G needle (5-mm depth), and (d) uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, real-time PCR, and analysis of proteoglycan (PG) content. Nerve ingrowth into the discs was assessed by immunostaining with the nerve marker protein gene product 9.5.ResultsInjured discs showed a progressive disc space narrowing with significant disc degeneration and proteoglycan loss, as confirmed by imaging results, molecular and compositional analysis, and histological examinations. In 16G punctured discs, nerve ingrowth was observed on the surface of scar tissue. In annular defects, nerve fibers were found to be distributed along small fissures within the fibrocartilaginous-like tissue that filled the AF. In discs filled with PLGA/ fibrin gel, more nerve fibers were observed growing deeper into the inner AF along the open annular track. In addition, innervations scores showed significantly higher than those of punctured discs and empty defects. A limited vascular proliferation was found in the injured sites and regenerated tissues.ConclusionsNerve ingrowth was significantly higher in PLGA/fibrin-filled discs than in empty defects. Possible explanations include (i) annular fissures along the defect and early loss of proteoglycan may facilitate the ingrowth process and (ii) biodegradable PLGA/fibrin gel may promote adverse growth of nerves and blood vessels into deeper parts of injured disc. The rabbit annular defect model of disc degeneration appears suitable to investigate the effects of nerve ingrowth in relation to pain generation.

Proteoglycan-depleted regions of anular-injury promote nerve ingrowth in a rabbit disc degeneration model

“…The consecutive levels of the rabbit IVD including L3/4, L4/5, and L5/6 were exposed by an anterolateral retroperitoneal approach. Annular injuries were randomly allocated to 4 disc levels: (1) Annular defects group: annular defects (diameter 1.8 mm; 4 mm depth) were created by a mini-trephine, as previously described [17,18]; (2) PLGA/ brin/PBS group: annular defects were lled with a PLGA/ brin gel plug loaded phosphate buffered saline; (3) PLGA/ brin/chABC group: annular defects were lled with a PLGA/ brin gel plug loaded chABC; (4) Intact group: the L2/3 disc served as uninjured control. Finally, the wound was closed in layers.…”

“…Control immunoglobulins consistently yielded negative results. The ingrowth of immunoreactive nerve bers in the specimen was scored using a previously described grading scale [18,40].…”

“…In previous work we showed that a minimally invasive annulotomy-induced rabbit model was well established, an orientated growth-permissive nerves growing into annulus defects during the degenerative process. Also, loss of proteoglycan at the injury site involved in IVD degeneration have been observed to facilitate the in ltration of blood vessels and nerve bers into disrupted disc tissue [17,18].…”

“…Chondroitinase ABC (chABC) is a well-studied bacteria-derived enzyme for being able to speci cally depolymerize the glycosaminoglycan (GAG) side chains for the induction of mild disc degeneration and favor axonal regeneration by reduction of proteoglycan in the injury site [19][20][21][22][23][24]. According to our previous works, PLGA/ brin constructs as delivery system have good performance on biocompatibility, safety, and biodegradability [18,25], also in promoting in tissue repair without compromising the function of incorporating proteins [26][27][28][29][30]. Therefore, as a potential platform for the carrier of chABC, it should be a simple and desirable alternative to achieve local delivery in a controlled manner as to cleavage of GAGs via digestion overcome CSPG-mediated inhibition at the injured site of the degenerated IVD.…”

Intervertebral disc damage models in organ culture: a comparison of annulus fibrosus cross-incision versus punch model under complex loading

“…Rabbit Both injury models led to disc degeneration and loss of GAG [30] Box-cut by scalpel (4.5 × 4.5 mm) Bovine Injured IVDs showed significant height loss and a decrease in GAG content [31] End plate burst fractures and end plate puncture with 18G needle…”